BACTERIOLOGICAL REVIEWS, Sept. 1970, p. 228-277Copyright @ 1970 American Society for Microbiology

Vol. 34, No. 3Printed in U.S.A.

Bacterial RibosomeMASAYASU NOMURA

Laboratory of Genetics, University of Wisconsin, Madison, Wisconsin 53706INTRODUCTION............................................................ 228

GROSS FUNCTION OF THE RIBOSOME..................................... 229

RIBOSOME CYCLING; SUBUNIT EXCHANGE .............. ................ 230

FINE STRUCTURE AND FUNCTION OF RIBOSOMES AND RIBOSOMALCOMPONENTS ........................................................... 234

Chemical Characterization of Ribosomal Proteins................................ 235

Separation and characterization of ribosomal proteins........................... 235

Stoichiometry of ribosomal proteins ....... ................................... 236Primary Structure of Ribosomal RNA.......................................... 238

Size and possible subunits.................................................. 238

Heterogeneity of rRNA.................................................... 239

Base sequence analysis of rRNA............................................. 240

Secondary Structure of rRNA in Isolated States and in Ribosomes.................. 240

RNA-Protein Interaction and the Internal Organization of Ribosomes 241

FUNCTIONAL ANALYSIS OF MOLECULAR COMPONENTS.................. 244

Reconstitution of Ribosomes.................................................. 244

Partial reconstitution....................................................... 244Complete reconstitution................................................ 246

Chemical Modification of Ribosomes and Ribosomal Components................... 249

Genetics of Ribosomes....................................................... 251

Mutations at str and ram loci............................................... 251

Interaction among ribosome mutations........................................ 254

Clustering of ribosomal mutations........................................... 255

MECHANISM OF ASSEMBLY OF 30S RIBOSOMAL PARTICLES IN VITRO.... 255

Conditions Necessary for Total Reconstitution.................................. 255

Kinetics of the Assembly and the Presence of Intermediates........................ 256

Sequential and Cooperative Nature of the Assembly.............................. 256

BIOSYNTHESIS OF RIBOSOMES............................................ 258

Biosynthesis of rRNA........................................................ 258

Template for rRNA synthesis ......... ...................................... 258Maturation of rRNA...................................................... 258

Regulation of rRNA synthesis............................................... 260

Biosynthesis of Ribosomal Proteins............................................ 261

Template for ribosomal protein synthesis...................................... 261

Ribosomal protein pool..................................................... 262

Assembly Process ........................................................... 263Kinetic analysis of the flow of RNA precursors into mature ribosomes............. 263

Analysis with metabolic inhibitors............................................ 264

Ribosome assembly defective mutants........................................ 266

CONCLUDING REMARKS .................................................. 268LITERATURE CITED......................................................... 268

INTRODUCTIONZamecnik and his co-workers first established

the central role of ribosomes (then called micro-somal ribonucleoprotein particles) in proteinsynthesis, and in addition discovered most ofthe components involved in in vitro protein-synthesizing systems, such as transfer ribo-nucleic acid (tRNA) and aminoacyl-tRNAsynthetases. (For a review of earlier work, seereference 361). However, messenger RNA(mRNA) had not been discovered at that timeso it was thought that ribosomal RNAs were

the templates for the proteins synthesized onthe ribosomes. Thus it was hoped that studieson the structure of ribosomes and ribosomalRNA would give some clue as to the mechanismof information transfer from genes to proteins.About 1957 the first systematic studies on theisolation and characterization of ribosomes wereinitiated, mainly by Watson's group at Harvardas well as by the group at the Carnegie Institu-tion in Washington (23, 262, 263, 326). Thesestudies were done on ribosomes from Escherichiacoil and they established the following basic

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information. (i) Ribosomes isolated and purifiedin the presence of 10-2M Mg+2 have a sedi-mentation coefficient of 70S. The 70S ribosomesdissociate, upon lowering Mg+2 concentration,into two components, 50S and 30S subunits(326, 327). (ii) The 50S and 30S ribosomalsubunits have a particle weight of 1.8 X 106and 0.85 x 10, respectively (327; see also 123).(iii) Both 50S and 30S subunits contain abouttwo-thirds RNA and one-third protein. (iv)The 50S subunit contains one molecule of RNA,23S ribosomal RNA (rRNA); the presence of onemolecule of 5S RNA was discovered later. The30S subunit contains one molecule of RNA, 16SrRNA (152).

Subsequent progress in the study of ribo-somes, however, lagged far behind progress inother areas of molecular biology. This waspartly due to the discovery of mRNA which de-valued the role played by ribosomes, and partlydue to the difficulties caused by the structuralcomplexity of ribosomes. For example, 30Ssubunits contain about 20 different proteins,and 50S subunits contain about 30 to 35 differentproteins. Nonetheless, it was soon evident thatthe detailed mechanism of protein synthesiscould not be elucidated without knowledge ofthe structure and function of ribosomes. More-over, an active role for ribosomes in the codon-anticodon recognition process was suggested(105). Thus, serious interest in the ribosome wasrevived, and the last several years have witnessedrapid progress in the study of ribosomes. In thisreview, we shall describe this progress andattempt to summarize our present knowledge ofthe structure, function, and biosynthesis ofribosomes. We shall limit our discussion entirelyto bacterial ribosomes, and primarily to ribosomesfrom E. coli. We shall also focus our attention oncertain problems and discuss a few selectedpublished works rather than attempting to becomprehensive in surveying all the publishedpapers. Several reviews related to bacterialribosomes have appeared recently (154, 208,245, 281, 295, 297). In addition, topics relatedto ribosomes in mammalian cells have been re-viewed by Darnell (58).GROSS FUNCIION OF THE RIBOSOMEAlthough ribosomes may have several other

functions in vivo, e.g., stimulation of RNA syn-thesis or regulation of the biosynthesis of RNAor of ribosomes themselves, their only clearlyestablished functions are those related to thesynthesis of proteins. In this section we shallbriefly summarize our current knowledge of thevarious steps in protein synthesis which involvethe participation of ribosomes. The mechanism

of protein synthesis has been described in greaterdetail in several recent reviews (164a, 171).The initiation of protein synthesis requires

the formation of an initiation complex consistingof the 30S ribosomal subunit, mRNA andformyl-methionyl tRNA (112, 124, 232). Severalinitiation factors as well as guanosine triphos-phate (GTP) are required for this step. Initiationfactors are proteins which were originally ob-tained from crude ribosomes by 1 M NH4Clwashing and were found to be required for thetranslation of natural mRNA (68, 259, 307).At least three initiation factors, F1, F2, and F3(also called A, C, and B, respectively), are known(260, 343). The initiation site on the mRNAcontains an AUG codon which codes for fMet-tRNA (41). This was recently demonstrated bySteitz (308) and by Hindley and Staples (126),using the 70S initiation complex (see below)with RNA from an RNA phage as the mRNA.It is still not clear whether the selection of theinitiation site by the 305 ribosomal subunitscan occur without the participation of fMet-tRNA. Although one of the initiation factors(factor C or F2) has been shown to stimulatethe binding of 30S subunits to natural mRNAin the absence of fMet-tRNA (107, 121), it hasnot been established that this binding takesplace at the correct initiation site. The functionof the initiation factor F2 may be the generalstimulation of mRNA binding to 30S subunitsregardless of the kind of codon. Artificial initi-ation of polypeptide synthesis using acetyl-phenylalanyl-tRNA as the initiation tRNA andpolyuridylic acid (poly U) as the mRNA at alow Mg+2 concentration was also shown torequire the initiation factors (175). Moreover,the binding of poly U to ribosomes was shownto require the initiation factors under conditionsof low Mg+2 concentration (42).

After formation of the initiation complexconsisting of the 30S subunit, mRNA, fMet-tRNA, and the initiation factors, the 50S ribo-somal subunit joins to form the 70S initiationcomplex (95, 120, 233). GTP hydrolysis is be-lieved to take place at some step after the joiningof the 50S subunit (147). The fMet-tRNA isnow located on the P site of the ribosome whereit can react with puromycin, and the A site isleft free for occupation by the next aminoacyl-tRNA. Many biochemical studies have beendone on the initiation steps (5, 6, 216, 260, 270),but the details are still unclear.The next step is the binding of a second

aminoacyl-tRNA to the A site. This binding isdirected by the codon next to AUG and requiresGTP as well as two soluble protein factors, Ts

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and Tu (72, 173, 257). The T factors (Ts and Tu)interact first with GTP and then with an amino-acyl tRNA, with the exception of fMet-tRNAFand Met-tRNAF (242). The GTP-aminoacyltRNA-T factor complex then reacts with theribosome leading to binding of the aminoacyltRNA at the A site. GTP appears to be hydro-lyzed at this step (243). Although codon-directedaminoacyl tRNA binding to 30S ribosomal sub-units in the absence of 50S subunits was observedwith some aminoacyl-tRNAs other than fMET-tRNAF, most of the binding of aminoacyl-tRNAsrequires the presence of both 305 and 505 sub-units (232). It appears that the only physiologi-cally significant aminoacyl-tRNA binding to free30S subunits is that of fMET-tRNAF. The sta-bility of the mRNA-aminoacyl-tRNA-ribosomalcomplex has been studied by Grunberg-Managoand her co-workers (109). There was no signifi-cant difference between the stability of the 705ribosomal complex involving fMet-tRNAF andthe stability of the 70S complexes involving otheraminoacyl-tRNAs. However, the 305 ribosomalcomplex involving fMet-tRNAF was more stablethan 30S complexes involving other aminoacyl-tRNAs.The next step in protein synthesis is the forma-

tion of a peptide bond between fMet-tRNAF andthe second aminoacyl-tRNA bound to the ribo-some. This peptide bond formation does not re-quire any supernatant protein factor, and iscatalyzed by the 50S subunit (210, 339). Monrodeveloped a model system in which washed 50Ssubunits catalyze the reaction of a 3'-terminalfragment of fMet-RNAF (CAACCA-fMet) withpuromycin (210, 211). Thus, the enzyme respon-sible for this peptide bond formation (calledpeptidyl transferase) is an integral part of the 50Ssubunit. After formation of the first dipeptidebond, the formylmethionyl aminoacyl-tRNAstays at the A site, and the discharged tRNAFstays on the ribosome, probably at the original Psite.The next step is the so-called translocation step,

that is, movement of fMet-aminoacyl-tRNA (orpeptidyl-tRNA, in general) from the A site to theP site. Release of discharged tRNAF from the Psite accompanies this translocation step (151,174). Translocation requires a soluble proteinfactor, called the G factor, and GTP, which ishydrolyzed to GDP and Pi (71, 113). Simultane-ously with translocation, the ribosome movesalong the mRNA by the length of one codon,leaving the third codon ready for the binding of anew aminoacyl-tRNA to the A site. These pro-cesses are repeated and polypeptide chain elonga-tion continues until the ribosome encounters one

of the chain termination codons (UAG, UAA, orUGA). Chain termination leads to cleavage of thepolypeptide from the tRNA and its release fromthe ribosome.Chain termination requires the participation of

release factor proteins in addition to terminationcodons, (36, 37, 39, 284; see also 38, 199, 283).Two release factors, R1 and R2, are known. R1 isrequired for the termination codons UAG andUAA, and R2 is required for UAA and UGA.For termination to take place, the peptidyl-tRNAmust be on the P site (37, 38) and both 30S and50S subunits are required (38). The state of ribo-somes following chain termination is controversialand will be discussed in the next section.Thus ribosomes appear to have many functions

in protein synthesis. First, they must recognizethe initiation site on mRNA. Second, they mustprovide sites for the binding of various molecularcomponents, such as mRNA, aminoacyl-tRNAs,initiation factors, several transfer factors, andrelease factors, so that these components caninteract with each other in a precisely orderedway. Third, ribosomes must move along themRNA during the translocation reaction. Fourth,they must catalyze the peptidyl transferase re-action. In addition, ribosomes are known toinfluence the accuracy of the codon-anticodonrecognition reaction.One major feature of the ribosome structure,

the presence of the 30S and the 50S subunits, wasinitially difficult to explain. With the large varietyof ribosomal proteins, it was thought that all theribosomal functions could be performed on asingle particle. However, the initiation theoryascribing a specific role to the 30S subunit makesdissociation of the 70S ribosome into 30S and50S subunits a property that is obligatory forinitiation and that may also serve as a regulatorymechanism. Other models in which the subunitconstruction was used to explain the efficient per-formance of the translocation reaction were alsoproposed (26, 296). However, these models arestill speculative and are difficult to test experi-mentally. Clearly, one of the major purposes instudying ribosomes is to clarify their structuralorganization and to understand how the functionsdescribed above can be performed efficiently.

RIBOSOME CYCLING; SUBUNITEXCHANGE

The model of chain initiation proposed byNomura and Lowry (232) has a direct and readilytestable consequence: a given 70S ribosome mustdissociate to start protein synthesis. That is, agiven 705 ribosome will not be conservedthroughout its functional lifetime, but will peri-

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odically dissociate, reforming from a subunit poolat each new cycle of initiation and translation.The concept of such a "dynamic" 70S was put

forth in 1964 by Watson, when he considered thesignificance of the universal presence of two un-equal ribosomal subunits in relation to ribosomefunction (347a). Two years later the theory wasadvocated and revitalized by Mangiarotti andSchlessinger (181). Using a new method of celllysis, they observed a ribosomal profile containingpolysomes and ribosomal subunits in roughlyequal amounts. They found no 70S ribosomes.Owing to the speed with which they were able tocarry out cell lysis and the fact that their condi-tions were at least gentle enough to preserve alarge number of polysomes, they concluded thatthe distribution was representative of the in vivosituation. The ribosomal subunits in their ex-tracts do not form 70S even in the presence of ahigh concentration of Mg+2 (0.01 M), and do notcarry pulse-labeled nascent peptide. Such ribo-somal subunits had been known as "native"ribosomal subunits and were originally describedby Green and Hall (106). Mangiarotti and Schles-singer thus suggested that 70S ribosomes ob-served in cell extracts by previous workers areformed through degradation of polysomes; thecompletion of translation yields 30S and 50S sub-units which rejoin at the next cycle of initiation.Although these observations were at least con-

sistent with the proposed ribosome life cycletheory, it was clear that direct evidence wasneeded before the theory could be accepted asfact. Moreover, their crucial observation, theabsence of 70S ribosomes in vivo, was not ac-cepted by several other investigators who ob-served 70S ribosomes in extracts prepared byequally gentle methods. The direct evidence forthe cyclic dissociation of ribosomes was providedlater by the elegant experiments of Kaempfer,Meselson, and Raskas (137). Using the techniqueof density labeling, they showed the rapid forma-tion of 70S ribosomes with hybrid density aftershifting a culture of E. coli from a heavy medium(containing 'IC, 15N, and 2H) to a light one (con-taining 12C, 14N, and H). Two types of hybrid 70Sribosomes were demonstrated, one consisting ofheavy 50S and light 30S, and the other consistingof light 50S and heavy 30S ribosomal subunits.It was also shown that both 50S and 30S subunitsare stable and remain intact during growth. Thedata suggested that all ribosomes are subject tosubunit exchange and that this exchange probablyoccurs many times within a single bacterialgeneration.Once the fact of subunit exchange during

bacterial growth was established, it was then

necessary to establish a causal relationship be-tween cyclic subunit exchange and the cycle ofprotein synthesis. The subunit exchange mightreflect some other cellular functions. For example,it is conceivable that the observed subunit ex-change reflects a mechanism of ribosome bio-synthesis; biosynthesis of a new 70S ribosomeparticle might necessitate its dissociation intosubunits to act as "catalyzers" or "organizers" tofacilitate assembly of the other subunits. That thecyclic subunit exchange is in fact causally relatedto the successive rounds of translation wasdemonstrated by Kaempfer (135). He showedrapid subunit exchange in in vitro incubationmixtures containing differentially labeled heavyand light extracts. Several agents known to blockprotein synthesis, such as sparsomycin and tetra-cycline, were found to prevent the formation ofribosomes with hybrid density. Furthermore,although for technical reasons it was not possibleto determine the rate of exchange in vivo, Kaemp-fer could observe considerable hybrid formationin vitro within 20 sec of incubation. From theseresults he concluded that ribosomes probablyundergo subunit exchange after each round oftranslation.

It was necessary to prove that this apparentdependence of subunit exchange upon proteinsynthesis was the direct result of the mechanism ofchain initiation as originally proposed by Nomuraand Lowry (232): the selective binding of fMet-tRNA to the 30S subunit and not to the 70Sribosome.

In 1968, Guthrie and Nomura (112) demon-strated that such was the case. Using 70S ribo-somes labeled with heavy isotope, the binding offMet-tRNA and val-tRNA in response to theappropriate synthetic messenger (random polyAUG or poly UG) was followed in the presenceof excess light 50S subunits. Analysis of the reac-tion products revealed that, whereas fMet-tRNAwas bound preferentially to the hybrid ribosomesconsisting of heavy 30S and light 50S subunits,val-tRNA was bound almost exclusively to theheavy, or conserved, ribosomes. Thus, whereas anoninitiation tRNA gan only be bound directlyto a 70S ribosome, the 70S ribosomes whichunderwent the subunit exchange bound the initia-tion tRNA to the exclusion of any other tRNA.It was thus concluded that the binding of fMet-tRNA to 70S ribosomes involves formation of theinitiation complex (30S-mRNA-fMet-tRNA com-plex) as an obligatory intermediate. Since all the"normal" protein synthesis in bacterial extractsstarts with fMet-tRNA, these experiments for-mally prove that all protein synthesis in bacteriastarts on the 30S subunit. Thus this work, to-

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gether with the experiments of Kaempfer andMeselson, convincingly showed the general va-lidity of the ribosome life cycle outlined in Fig. 1.

In vivo experiments performed by Mangiarottiand Schlessinger (182) using 3H-uracil pulse-labeling techniques showed that the specificactivities of free ribosomal subunits and thosepresent in polysomes increase at identical rates.This is consistent with very rapid subunit ex-

change during protein synthesis. Ghosh andKhorana (95) observed the stepwise formation ofthe dipeptide fMet-met in vitro. Starting with free30S subunits, they bound fMet-tRNA in response

to the repeating polymer poly AUG and isolatedthe resultant complex. Addition of 50S subunits,met-tRNAM, and supernatant enzymes producedthe dipeptide. Thus the initiation of peptide bondformation can occur starting with the 30S initia-tion complex.Many other in vitro experiments have con-

firmed the mechanism of chain initiation startingwith 30S subunits (83, 108, 109, 124, 216, 260,269). The most recent experiment from Thach'slaboratory (120) is particularly pertinent. It was

originally found by Eisenstadt and Brawermanthat the initiation factors are present in the na-

tive 30S fraction and not in 50S subunits or 70Sribosomes (68a; see also 251). Thach and his co-

workers prepared 3H-labeled initiation factor F1and showed that the factor is incorporated intothe 30S initiation complex (30S-fMet-tRNA-AUG) but that it is released from the complexwhen the 50S subunit joins. These results de-lineate the step involving factor F1 and stronglysupport the 30S initiation complex as an obliga-tory intermediate in the formation of the 705initiation complex.One premise of the ribosome life-cycle theory is

that the native 30S and 505 subunits combineonly at the initiation site on mRNA. The predic-tion from the theory was that the subunits wouldcombine only under conditions that allow chaininitiation. Thus several workers examined theconditions necessary for the formation of 305-50S couples. Initially, Schlessinger, Mangiarotti,and Apirion (282) found that mRNA, tRNA, K+,and Mg+2 were required for 70S formation. TheMg+2 concentration needed was dependent on thenature of the mRNA used, 10 mm being sufficientwith poly AUG, whereas 20 mm was requiredwith a poly U template. A curious finding was

that stripped tRNA could function as well as

charged tRNA in stimulating couple formation.These experimental results supported the theoryin general, but were unsatisfactory in that theconditions were different from those thought tobe required for "natural" chain initiation. More

*MET MET

r mmRNA mRNA

(30S INIATION (70S INnATIONCOMPLEX) COMPLEX)

mRNAf MET-tRNAINITIATIONFACTORS

ETC.

(POLYSOME)

FIG. 1. Ribosome life cycle.

recently, Kondo and his co-workers (148) showedthat the association of 14C-30S and 50S nativesubunits in the presence of GTP, natural mRNA(f2 phage RNA), and initiation factors at 5 mMMg+2 requires the presence of fMet-tRNAF. Ifthe fMet-tRNAF is replaced by unchargedtRNAF, unformylated met-tRNAF, unfraction-ated tRNA, or purified phe-tRNAPhe, no 14C-70Sribosomes are formed. Under the conditionsused, there is no detectable binding of unformyl-ated met-tRNAF or of phe-tRNA to 30S sub-units. In this way, it was demonstrated that thecoupling reaction is specifically due to the initia-tion event.

It is thus clear that the general validity of thetheory of a ribosome life cycle is firmly estab-lished. However, there are several unsolvedproblems regarding the details of the ribosomelife cycle. (i) The question whether ribosome dis-sociation takes place concomitant with chaintermination or via some free 70S ribosome inter-mediate has not been settled. (ii) We have so farassumed that there is no subunit exchange duringthe translation of a cistron in mRNA. From thestriking stimulation of in vitro subunit exchangeby puromycin, Kaempfer and Meselson (136)argued for the absence of extensive "intracis-tronic" subunit exchange during the normaltranslation process. It is desirable to prove theabsence of such exchange more rigorously. (iii)Most of the mRNAs in microorganisms appearto be polycistronic and to be read sequentiallyfrom the first cistron. Thus, the question ariseswhether the ribosomes dissociate at the chaintermination signal in the middle of a polycistronicmRNA, as our current knowledge of the initia-tion mechanism would seem to require.With regard to the first question described

above, it was originally proposed that ribosomedissociation takes place concomitantly with chain

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termination. Two observations made by Schles-singers's group led to this conclusion: (i) theabsence of 70S ribosomes in gently preparedbacterial extracts (181); (ii) the dissociation ofribosomes in vitro concomitant with the peptidylchain release occurring after puromycin treat-ment (282). However, neither of these observa-tions was confirmed by other investigators andthe conclusion was seriously challenged. First, asignificant number of 70S ribosomes was ob-served by other workers in gently preparedbacterial extracts (2, 98, 141, 146). These workersargued that the observed 70S ribosomes are notbreakdown products of polysomes. The majorevidence is as follows. (i) Several specific varia-tions of in vivo conditions before cell breakagealtered the number of 70S ribosomes, even thoughthe same methods of cell breakage and extractionwere used. Thus, slow cooling of cells (98), cellaging (254, 255), amino acid or carbon sourcestarvation, and actinomycin D treatment (146)all caused breakdown of polysomes and increasedthe number of 70S ribosomes, but not the numberof 30S and 50S subunits. All of these conditionswere thought to reduce protein synthesis slowlyand to cause the accumulation of 70S ribosomesas the run-off products of mRNA translation.(ii) Comparison of the stability of "complexed"70S (70S obtained from the ribonuclease treat-ment of polysomes) and "free" or "run-off" 70S(obtained from amino acid-starved cells, forexample) at various Mg+2 concentrations showeda clear-cut difference: "complexed" 70S ribo-somes, like polysomes, were more stable than"free" 70S ribosomes, and required a greaterreduction in Mg+2 concentration for dissociation(141, 265). (iii) Peaks containing predominantly"free" 70S ribosomes were shown to have a muchsmaller number of growing peptides and lessmRNA than "complexed" 70S ribosomes de-rived by fragmenting polysomes (2, 265). Withrespect to puromycin, Davis and his co-workers,in extensive studies, showed that puromycincauses the release of growing peptide chains, invivo and in vitro, without causing 70S dissocia-tion (146); that is, they failed to confirm Schles-singer's original experiments. Thus, both of theoriginal observations used for the "direct dis-sociation hypothesis" were seriously challenged,and 70S ribosomes were claimed to be the directrun-off products (Fig. 2B).Although the published experimental data are

sometimes conflicting and the general picture isstill confusing (see papers cited above, and also54, 83, 254, 255), it now appears that "free" 70Sribosomes, as distinguished from polysome break-down products, do exist in extracts. A major

question remains as to whether they actuallyexist in vivo or are formed during or after celllysis. Phillips and Franklin (254) suggested thatthey are mainly in vitro artifacts. They observedtwo types of ribosome distribution in bacterialextracts, depending on the type of monovalentcation present in the lysing medium. The patternwith a high proportion of 70S was obtained in K+or NH+4 media and that with a low proportionof 70S in a Na+ medium. They also found thatthe addition of tetracycline to lysates prepared inK+ buffer resulted in a 40% decrease in the num-ber of 70S ribosomes. It is known that K+ orNH+4, but not Na+, is required for the bindingof aminoacyl-tRNA to ribosomes and that suchbinding is inhibited by tetracycline (122, 312).These observations suggested that 70S ribosomesare formed from 30S and 50S subunits in vitro asa result of aminoacyl-tRNA binding. However, itmust be remembered that the mode of action oftetracycline is still only incompletely understood.Phillips and Franklin also failed to show theformation of 70S from native 30S plus 50S sub-units in the K+ or NH+4 media in vitro. RecentlyBeller and Davis (personal communication)showed that the Na+ medium causes dissociationof free 70S ribosomes, and they interpreted thedata of Phillips and Franklin on that basis. Thusthe available data favor the possibility of theexistence of 70S ribosomes in vivo.

If free 70S ribosomes are not in vitro artifacts,three possibilities can be considered: (i) appar-ently "free" 70S ribosomes are some kind ofinitiation monosome which has mRNA and isstabilized by fMet-tRNA or some other tRNAderivative; (ii) free 70S ribosomes are not di-rectly involved in the ribosome cycle, but are ona side pathway as shown in Fig. 2A; and (iii) free70S ribosomes are the direct run-off products ofpolysomes (Fig. 2B).

Concerning the first possibility, Davis and hisco-workers (146) noted the absence of formyl-methionine in their free 70S ribosomes. The sameobservation also comes from experiments done byPhillips and Franklin (254). When they treatedcells in vivo with trimethoprim, which leads to adeficiency of fMet-tRNA, they noted a great de-crease in polysome content. This was concomi-tant with an increase in 70S particles in K+lysates or an increase in free 30S and 50S in Na+lysates. These observations suggest that fMet-tRNA is not responsible for the formation orstabilization of 70S ribosomes in K+ or NH+media. Thus, the first possibility is unlikely.As to the second and third alternatives, the

accumulation of 70S ribosomes under variousconditions, especially after puromycin treatment,

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A

+ 50530S-initiation complex

30I 30S + 505 pool

705

B

I+'

,705-initiation complex - Polysomes

I Chain termination

30S + 50S

complex Polysomes505u-initiation5Sot

OS-initiation complex I Chain ter

30ST a 30s + 50S pool 147e0SFIG. 2. TWO alternative models for the role of "free" 70S ribosomes in the ribosome cycle in vivo.

was taken by several workers as strong supportfor free 70S ribosomes being direct run-off prod-ucts. However, Kaempfer and Meselson (136)showed very recently that treatment of a mix-ture of heavy and light extracts with puromycincauses the very rapid accumulation of hybrid 70Sribosomes. This experimental result implies thatthe free 705 ribosomes accumulated after puro-mycin treatment are not the direct run-off prod-ucts of mRNA translation. It strongly suggeststhat the run-off products are 50S and 305 sub-units, which then rapidly reassociate. Althoughwe cannot yet decide whether scheme 2A or 2B(Fig. 2) is correct, both require the dissociationof the 70S before it can be reutilized for chaininitiation. If free 70S ribosomes really do existin vivo, then their dissociation must somehowbe effected and controlled as suggested by Sub-ramanian et al. (313). A factor which causes dis-sociation of free 70S ribosomes, but not 70Sderived from polysomes, was isolated by Davisand his co-workers and subsequently by others(17, 313). Recent studies (313a) suggest thatthe dissociation factor is identical to one ofthe initiation factors, F3, studied by previousworkers. Although the nature and significance ofsuch a factor must await further studies, it couldfunction to regulate the level of free ribosomalsubunits available for the initiation of proteinsynthesis.

It appears from the foregoing discussion thatwe are not in a position to make any definitiveconclusions as to the direct products of chaintermination or the nature of free 70S ribosomes.It would seem that, as in the case of initiation, aconvincing answer could be obtained by re-

sorting to simpler in vitro systems, such as thatdeveloped by Capecchi (36). As to the nature of

free 70S ribosomes, rigorous purification andcareful biochemical studies would be required toidentify the factors responsible for uniting thetwo subunits. Although such studies may needpainstaking effort and may not be directly relatedto the primary question about the chain termina-tion products, they should certainly provide use-ful information related to structural and func-tional aspects of ribosome subunit interactions.

FINE STRUCTURE AND FUNCTION OFRIBOSOMES AND RIBOSOMAL

COMPONENTSSince about two-thirds of the ribosomal mass

is composed of rRNA, and since as much as 80%of the total cellular RNA is rRNA, it has beennatural to ascribe an important role to rRNA.This tendency, as well as the general hope offinding a simple structure for the ribosome, hasled many investigators to imagine the ribosomeas a particle consisting of functionally importantrRNA and a few proteins whose sole function isthe structural one of holding rRNA in a properconfiguration. This was perhaps one of thereasons why the heterogeneity of ribosomal pro-teins, though discovered as early as 1960 byWaller and Harris (347), was not immediatelyaccepted and why studies on the ribosomal pro-teins lagged far behind studies on rRNA untilquite recently. As I shall describe below, recentdevelopments in this field have clearly shown thatthere are many chemically and functionallydifferent kinds of ribosomal proteins, and we are

now obtaining considerable information abouttheir functional significance, especially thosefrom the 30S ribosomal subunit. On the otherhand, despite the large amount of work on rRNA,

rmination

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our knowledge of its structure and function isstill very limited.

Chemical Characterization of RibosomalProteins

The pioneer work done by Waller and Harris(346, 347) showed that ribosomes contain manydifferent kinds of proteins. This was a surprisingresult, since earlier models for the ribosome com-pared it to simple viruses which have proteinshells consisting of many copies of one or a fewkinds of proteins. Waller solubilized ribosomalproteins with acetic acid and then showed thepresence of many protein bands after starch gelelectrophoresis. Because the electrophoresis wascarried out in the presence of urea, the multi-plicity of proteins revealed could not be a result ofaggregation. Waller found that 30S and 50Sparticles show different protein compositions andthat different bacterial species show differentprotein patterns.Although Waller's chemical work did not rigor-

ously prove the chemical heterogeneity of riboso-mal proteins, it nevertheless provided very strongexperimental evidence for this notion and stimu-lated other investigators to undertake the formi-dable task of the separation and characterizationof each of the many ribosomal proteins. Twoother methods of approach yielded results con-firming those of Waller and indicated the hetero-geneity of ribosomal proteins before more com-prehensive chemical work was undertaken. Onewas the demonstration of a chromosomal locuswhich controls the characteristic electrophoreticbehavior of one of the many proteins (K protein)appearing in the polyacrylamide gel electropho-retic pattern (162). The other was the demonstra-tion that the controlled degradation of ribosomesby a high concentration of CsCl yields proteinfractions which have specific functions in the re-constitution of ribosomes and correspond to sub-sets of the group of ribosomal proteins nor-mally displayed in a polyacrylamide gel electro-phoretic pattern (129, 305, 334).

Separation and characterization of ribosomalproteins. Most ribosomal proteins are basic and,as a mixture, are insoluble in ordinary buffers atlow salt concentrations. Several methods havebeen used to extract and solubilize these proteins.Acetic acid (66%) was used first by Waller andHarris (346, 347). The urea-LiCl method wasdeveloped by Spitnik-Elson (300) and used bysome other groups (162, 331). 2-Chloroethanolwas used by Fogel and Sypherd (80). Digestionby ribonucleases in the presence of urea was alsoused (247, 290, 299). It is certainly possible thatdifferent extraction methods yield protein prep-

arations somewhat different from each other.This should be kept in mind whenever one ob-serves some differences in the results obtained byvarious investigators.The extracted proteins have been separated and

purified by column chromatography on carboxy-methyl cellulose, phosphoceilulose or DEAEcellulose, and by preparative polyacrylamide gelelectrophoresis, by gel filtration, or by a combina-tion of these methods. Because of the insolubilityof ribosomal protein mixtures in dilute salt solu-tions, these separations were usually carried outin the presence of urea. Since the number ofribosomal proteins is very high, 30S and 50Sribosomal subunits were first separated by zonalor fractional centrifugation. Also, crude groupfractionation methods, such as centrifugation ofribosomes in high salts (329, 337) or fractionalprecipitation of extracted proteins with salts, weresometimes used as a preliminary step before theabove-mentioned separation methods were used.With the methods described above, Kalt-

schmidt and his co-workers first purified 22 pro-teins from E. coli 70S ribosomes and studied theiramino acid composition, tryptic peptides, andmolecular weight (140). Moore and Traut andtheir co-workers at the University of Genevapurified 13 proteins from 30S subunits (213, 340)and showed that these proteins were distinct fromeach other with respect to their amino acid com-position, tryptic peptides, and molecular weight.These experiments established convincingly thatthe multiple components on gel electrophoresiscorrespond to different protein molecules. Thesame conclusion was also reached by Fogel andSypherd (81), who studied 14 proteins purifiedfrom 30S ribosomal subunits. Subsequently, morecomprehensive and careful work on the enumera-tion and characterization of 30S proteins waspublished by Kurland and his coworkers (53,115).

Before we describe these chemical results indetail, some problems involved in the identifica-tion of ribosomal proteins should be discussed.When a purified protein preparation is obtainedfrom ribosomes, we encounter three kinds ofproblems before we can classify it as a genuineribosomal protein. First, the protein may be anonribosomal protein which is bound to theribosome. An example is ribonuclease I, whichexists outside the E. coli membrane in vivo butbecomes bound exclusively to the 30S ribosomalsubunit when the bacterial cells are broken withmethods commonly used (228). Second, the pro-tein in question may be an artifact produced bychemical or enzymatic modification of some otherprotein during the course of extraction and pun-

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fication. Third, the given protein preparation maynot be pure, consisting of a mixture of two ormore proteins.The first problem is a difficult one and is related

to the definition of ribosomes. A common ap-proach is to purify the ribosomes rigorously with-out decreasing their functional activity in a cell-free protein-synthesizing system and to regardproteins contained in such ribosome preparationsas ribosomal proteins. For example, diethyla-minoethyl chromatography was used to get a very"pure" ribosome preparation (87). Kurlandpurified his ribosomes by (NH4)2SO4 precipita-tion (153), and obtained a lower protein contentthan that of preparations more commonly used.Other workers used washing through sucrosesolution or washing with a high concentration ofNH4C1. However, these purification methods maystill not be able to remove some tightly boundcontaminating proteins. It should also be pointedout that some proteins, such as the initiationfactors, are removable by high-salt washings andyet are required for certain ribosomal functionsand could be defined as ribosomal proteins.(However, the initiation factors are usually notclassified as ribosomal proteins.)Another criterion for a ribosomal protein is

the demonstration that its presence is necessaryfor obtaining functional ribosomes in an in vitroribosomal reconstitution system, such as the onedeveloped in my laboratory (331). Of 21 pro-teins purified by us, 19 were shown to be func-tionally necessary by this method. The thirdcriterion is genetic. When a protein in a ribosomalprotein preparation is shown to be altered as aresult of a mutation affecting a presumed ribo-somal function, the protein in question can beconsidered a genuine ribosomal protein. Thus,both the 30S protein P10 controlled by the strlocus (19, 250) and P4 controlled by the spc locus(22) can be regarded as genuine ribosomal pro-teins.The second problem, the possibility of a given

protein being an artifact, was considered andeliminated by several workers for most of theisolated proteins. Thus, aggregation, deamination,carbamylation, disulfide bond formation, andproteolytic degradation were shown not to beresponsible for the production of most of theproteins purified (115, 207, 336, 346).The third problem, that of the purity of a given

protein preparation, is usually examined byphysical methods Most commonly used is poly-acrylamide gel electrophoresis (in the presence ofurea or sodium dodecyl sulfate) at several differ-ent gel concentrations (80, 162, 337). More care-ful examination was done by Craven et al. (53),

who calculated the chemical molecular weights ofthe ribosomal proteins from their amino acidcompositions and from the number of peptidesobtained in a tryptic digest. They compared thesevalues with those obtained by physical methods.With most of the 30S proteins, the two valuesshowed fairly good agreement. This suggests thateach of the proteins examined contains a singlemajor component.

It appears that the 30S ribosomal subunitcontains about 20 proteins. Craven and his co-workers concluded that the number of 30S pro-teins from E. coli B is between 19 and 22 (53),whereas the most recent work done by Traut andhis co-workers (337) lists 20 proteins from 30Ssubunits of E. coli MRE600. Amino acid composi-tions, tryptic peptides, and molecular weights ofthese proteins were studied (compare Table 1).In our own laboratory, 21 proteins were isolatedfrom E. coli K-12 strain Q13, and 19 of themwere found to correspond with proteins isolatedby Kurland's group (compare Table 1).

Traut and his co-workers (337) isolated 36proteins from 50S subunits and concluded thatthe number of 50S proteins could be between 34and 38. Kurland and his co-workers concludedthat the number of 50S proteins is between 25and 31 (Kurland, personal communication, see154). It is significant to note that there is no pro-tein common to both 50S and 30S subunits. Thisconclusion was obtained by both chemical (154,311a, 337) and immunological studies (337;Stoffler and Wittmann, personal communication).The question as to whether there is structural

similarity among these isolated ribosomal pro-teins was examined by several workers. AlthoughKaltschmidt et al. (140) reported some similaritiesin peptide maps of several ribosomal proteins,later immunochemical work done by the samegroup revealed very few cross-reactions amongthe ribosomal proteins (Stoffler and Wittmann,personal communication). Traut and his co-workers (337) examined tryptic peptides ob-tained from 13 pure 30S proteins and 12 pure50S proteins and failed to find any common pep-tide with the exception of some dipeptides. Theyalso used immunochemical methods to find pos-sible similarities among ribosomal proteins.Antisera to each of three purified 30S proteinswere shown to react only with the homologousproteins and not with any other 30S ribosomalproteins. These results favor the conclusion thatthere is no extensive structural homology amongribosomal proteins.

Stoichiometry of ribosomal proteins. It hasusually been assumed that the ribosome has adefined structure and that the ribosome popula-

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TABLE 1. Molecular weights and nomenclature of30S ribosomal proteins

Kurland's code Nomura's code Mol wte

1 P1 65,0002 Pa. 18,0002a P4b 17,6003 P4 24,0004 P6 16,0004a P2 30,0005b _b 31,0006b P9 (?)b 13,5007b P9 (?)b 10,7008 P5 21,5009 P3 33,00010 P4, 26,70011 P7 18,30012 P8 21,00012a P12 14,60012b P11 15,60013 Pl3 15,00014 Ploa 13,20015 P1O 19,00015a P15 13,00016 P14 14,000

P3bb

aData from Craven et al. (1969).b Identification of these proteins is not certain.

tion is homogeneous. The heterogeneity of ribo-somal RNA and hence of ribosome populationswas suggested by several investigators, but it isonly now that Kurland s group performed care-ful studies on the stoichiometry of ribosomalproteins that the question has been brought toserious consideration.

After the model with many identical subunitswas discarded and the reality of the existence ofmany protein bands on gels was established, itwas expected that each ribosome has one or twocopies of each of all the ribosomal proteins. Thus,when the first experimental studies on this prob-lem by the group at the University of Geneva(213) suggested that most (thirteen) of the ribo-somal proteins exist in amounts corresponding toone copy per 30S particle, it was readily acceptedthat the ribosomes are homogeneous with re-spect to their protein composition. However,later work by Kurland et al. (157) did not agreewith this conclusion. They examined 14 ribo-somal proteins and found that 6 of them arepresent in amounts corresponding to about onecopy per 30S particle, but that 6 other proteinsare present in amounts less than 0.7 copy perribosome. The remaining two proteins gave inter-mediate values. They concluded that there aretwo kinds of proteins; the class which they called

"unit proteins" is present in all the isolated 30Sribosomal particles, and the class which theycalled "fractional proteins" is present in somebut not all of the isolated 30S particles. Discrep-ancies between results obtained by Kurland'sgroup and those by the Geneva group are mostlyin the molecular-weight values assigned to someof the proteins. Although work done by anothergroup also favored the conclusion that all theribosomal proteins exist in stoichiometricamounts (81, 319), it now appears that the con-clusion obtained by Kurland's group is correct,at least with respect to ribosomes obtained as invitro preparations. Recent investigation of mo-lecular weights of 30S proteins by the group atthe University of Geneva has now yielded dataconsistent with that obtained by Kurland andhis collaborators (337). There are several otherfacts supporting the conclusion of a hetero-geneous population of ribosomes. First there is astriking correlation between proteins classified asunit proteins by Kurland et al. and proteinsfound by us to be required for the "physical as-sembly" of ribosomes (see Table 3). Such acorrelation is consistent with the mechanism ofthe ordered assembly of 30S particles discussedbelow and it is difficult to believe that the cor-relation is fortuitous.

Second, Kurland and his co-workers were ableto show as much as 60% stimulation of activityof 30S particles by incubating them with exter-nally added 30S proteins under the conditionsoptimal for reconstitution. Concomitant withthis stimulation, they observed that some ex-ternally added proteins were incorporated intothe particles and some proteins initially presentin the 30S particles were released into the medium(157). Although interpretation of the observedprotein exchanges must await exact identificationof these exchanged proteins, the observed factsare consistent with the conclusion that the iso-lated 30S particles are not fully active and thatpart of the reason for the inactivity is a deficiencyin some ribosomal proteins in some of the 30Sparticles.

There are several possible explanations for thenonstoichiometry of the 30S ribosomal proteins.(i) As discussed above, some proteins may benonribosomal proteins, such as ribonuclease Iwhich is tightly bound to the 30S particle. Pro-tein P1 was found to exist to the extent of only0.1 copy (157) or 0.4 copy (337) per ribosome.This protein was found to be not required eitherfor assembly or functional activity in the ribosomereconstitution assay (234; compare Tables 2 and 3and the section on Reconstitution of Ribosomes).Thus, it is very likely that P1 is a nonribosomalprotein. However, the explanation cannot be

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applied to proteins such as Pi, which are essentialfor 30S ribosomal functions (234, 329) and yetare reported to be present only to the extent of0.4 to 0.6 copy per ribosome (157; see Table 3).(ii) Some proteins may be lost during preparationof ribosomes and may exist in stoichiometricamounts in vivo. Although Kurland and co-workers showed that there are few losses duringpreparation of ribosomes from crude bacterialextracts with respect to most of the 30S proteins(157), the possibility cannot be excluded thatsome proteins are lost during cell breakage. Asignificant fraction of the ribosomes mav exist inclose association with cellular membranes invivo. It is conceivable that some of the basicribosomal proteins can easily be removed fromthe ribosome by forming insoluble aggregateswith acidic polymers in the cell membrane orwall. These proteins are then discarded as insol-uble cell residues during the preparation of cellextracts. (iii) The observed heterogeneity ofisolated ribosomal populations may reflect a realheterogeneity of ribosomal populations in vivo.Two models for heterogeneity (157) can be

considered. One involves permanently differ-entiated classes of ribosomes having somewhatdifferent functions. The second model assumesthat fractional proteins exchange from one 30Sparticle to another during the cycle of proteinsynthesis, depending on the functional require-ments imposed on the ribosome structure. Ac-cording to the latter model, the fractional pro-teins behave like some initiation factor proteinswhich exist bound to free 30S ribosomal subunitsbut are not present in the 70S structure of poly-somes (68a, 120, 251). Kurland et al. have usedthe in vitro protein exchange experiments de-scribed above as a support for this second model(157). However, the model needs more rigorousexperimental tests. For example, the proposedin vivo exchange of fractional proteins is in directconflict with the conclusion obtained by Kaemp-fer et al. (137). From their density-transferexperiments, Kaempfer et al. concluded that lessthan 2% of the material of the 50S subunit andless than 4% of the 30S subunit exchange duringbacterial growth; that is, both ribosomal subunitsremain intact or almost intact throughout manycycles of the translation process. Since the densityof ribosomes is determined mostly by RNA, it ispossible that the exchange of some ribosomal pro-teins may not have been detected. Experimentsspecifically designed to detect the proposed pro-tein exchanges in vivo should be performed totest this model. Another approach to proving thepostulated heterogeneity of ribosomal popula-tions is to fractionate the ribosomes and to show

functional and chemical differences among classesof the population. For example, immunochemicalmethods could be used to fractionate the popula-tion. Alternatively, different types of 30S ribo-somal particles could be isolated from cells de-pending on the functional state of the ribosomesin vivo. The protein composition of free (or"native") 30S subunits (106) may be differentfrom that of the 30S subunit in the 70S monomeraccumulated in vivo or in polysomes activelysynthesizing proteins.

In contrast to the 30S proteins, most of the 50Sproteins appear to be present in amounts cor-responding to one copy per 50S particle. Trautet al. (337) found that 31 of the 50S proteinsexist in stoichiometric amounts and only 2, orpossibly 4, of the 50S proteins exist in amountsthat are much less than one copy per particle.Kurland s group also failed to detect any signifi-cant heterogeneity of 50S ribosome populationsso far (Kurland, personal communication).A major conclusion which has emerged from

studies on ribosomal proteins is that, becausenone of the proteins has more than one copyper particle, ribosomal particles have no sym-metry. This means that any model of ribosomefunction involving structural symmetry, forexample the presence of two or more identicalsites on a ribosome, can be discarded.

Primary Structure of Ribosomal RNASize and possible subunits. It is the current be-

lief that the 30S ribosomal subunit contains onemolecule of 16S rRNA with a molecular weight of5.5 x 105, whereas the 50S ribosomal subunitcontains one molecule of 23S rRNA with amolecular weight of 1.1 x 106 and one moleculeof 55 RNA with a molecular weight of 4 X 104(31, 152, 306). Since the molecular weight of 23SrRNA is about twice that of 16S rRNA, therehave been frequent claims that the 23S rRNA is adimer of a "16S" RNA molecule which is identi-cal or very similar to the 16S rRNA moleculefound in 30S ribosomal subunits. Several observa-tions supported this idea. (i) It was originallyfound that RNA with a sedimentation behaviorvery similar to the 16S rRNA of 30S subunitscould be isolated from 50S subunits together with23S rRNA (152). (ii) The specific conversion of23S rRNA into 16S RNA was observed under avariety of conditions (196). (iii) Midgley esti-mated the chain length of 23S rRNA from thenumber of 3'-terminal bases measured by NaIO4oxidation followed by '4C-isonicotinic acidhydrazide treatment. Although he originally re-ported a chain length of about 3,100 for the 23SrRNA (195), which is consistent with the meas-

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ured molecular weight of 1.1 X 106, he andMcIlreavy later observed that the chain length of"rigorously purified". 23S rRNA is 1,500 (190),and they claimed that the 50S ribosomal particlecontains two RNA chains, each correspondingto a molecular weight of about 5 X 105. (iv)Finally, recent work done by Fellner and Sanger(76) showed that all the methylated oligonucleo-tides obtained after T1 ribonuclease digestion of23S rRNA are present in an amount correspond-ing to 2 moles of each particular oligonucleotideper mole of 23S rRNA with molecular weight of1.1 X 106.We can now exclude the claim that 23S rRNA

is a dimer of 16S rRNA identical to the 16S rRNAderived from 30S ribosomal subunits. The 23Sand 16S rRNAs have been shown to be differentin base composition (306), base sequence asjudged by deoxyribonucleic acid (DNA)-RNAhybridization (14, 180, 239, 287, 357), oligonu-cleotide patterns obtained after enzymatic diges-tion (10), methylated oligonucleotides obtainedafter T1 ribonuclease digestion (76), and in their5' terminal sequence (321). It was also found thatthe "16S" RNA prepared from 23S rRNA ac-cording to the method of Midgley (196) cannotreplace 165 rRNA in the reconstitution of 30Sribosomal subunits (331; see FUNCTIONALANALYSIS OF MOLECULAR COMPO-NENTS). Thus, it is unlikely that the 23S rRNAis formed by simple dimerization of two 16SrRNA molecules.The claim that the 50S ribosomal subunit con-

tains two RNA chains (plus 5S RNA) is alsodifficult to accept. First, the conversion of 23SrRNA into smaller RNAs which had been claimedby several workers could not be observed underconditions minimizing nuclease contamination(206). Second, careful studies by Stanley andBock (306) revealed no noncovalent bond in the23S rRNA molecule. Finally, Leppla (165)measured the number of chain terminal bases inthe 23S rRNA molecule by a method similar tothat used by Midgley. Leppla used NaIO4 oxida-tion followed by 3H-labeled NaBH4 treatmentand obtained results which are consistent withone chain terminus per 1.1 X 106 daltons of 23SrRNA. Thus we are confident that the 23S rRNAis a single polynucleotide chain with a molecularweight of 1.1 X 106.

According to Marrs and Kaplan (186) "ma-ture" 50S ribosomal subunits of Rhodopseudo-monas spheroides contain two RNA species, 16SRNA and 14S RNA, and 23S RNA can be de-tected only by pulse labeling. They suggest that23S RNA is cleaved into two RNA species, 16SRNA and 14S RNA, during maturation of the

50S ribosomal subunit. This appears to be anexceptional case.

Although the 23S rRNA is a single polynu-cleotide chain, the work of Fellner and Sanger(76) strongly indicates that the molecule is madeup of two sections which may be identical or verysimilar in their base sequence. One of their sug-gestions is that the 23S rRNA cistron has arisenby a "gene duplication" mechanism duringevolution. Whether the possible existence of twoidentical or similar parts is related to the functionsof 23S rRNA (354) is not clear. A related sub-ject is the problem of sequence homology be-tween 16S and 23S rRNAs. Despite clear-cutevidence for a sequence difference between E.coli 16S and 23S rRNAs, DNA-RNA hybridiza-tion experiments have shown that 16S and 23SrRNAs compete for the same DNA sites to agreat extent (14, 180). This suggests that DNAcistrons for both 16S and 23S rRNA have evolvedby gene duplication starting from a commongene. Alternatively, partial sequence homologymay reflect a common (unknown) functionperformed by parts of both 16S and 23S rRNAs.On the other hand, DNA-RNA hybridizationexperiments done with Bacillus megaterium andB. subtilis showed a complete lack of sequencehomology (239, 287, 357). It is not clear whetherthe observed discrepancy is due to a difference intechniques used or to the difference in bacterialspecies. It is desirable that these experiments berepeated using identical experimental techniquesfor the two organisms.The 50S ribosomal subunit contains one

molecule of 5S RNA in addition to 23S rRNA(268). The 5S RNA does not accept amino acidsand thus is different from tRNA. It is not a ran-dom breakdown product of 16S or 23S rRNAbut appears to be a genuine ribosomal componentpresent in all 50S subunits of various origins.However, its functional role is totally unknown.The 5S RNA from E. coli consists of 120 nucleo-tides, and its base sequence has been completelyelucidated (31). No base sequence homology hasbeen found between 5S RNA and 16S or 23SrRNA by using the technique of DNA-RNAhybridization (362). Also, 5S RNA does notcontain any methylated or unusual bases, incontrast to other rRNAs or tRNAs (31).

Heterogeneity of rRNA. DNA-RNA hybridiza-tion experiments have clearly shown that genesfor 16S and 23S rRNA are present in multiplecopies (357), perhaps on the order of 10 copiesper bacterial genome. Because of this redundancy,it is quite possible that the genes for 16S rRNAor those for 23S rRNA are not homogeneousand that there are several chemically different

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species of 16S rRNA and 23S rRNA. The ques-tion of the possible heterogeneity of rRNAs isimportant, since it implies the heterogeneity ofeach of the ribosomal subunits and is possiblyrelated to some functional differentiation amongribosomes.There are several reports which suggest hetero-

geneity of 16S and 23S rRNA. First, Aronsonand Holowezyk (11) observed alterations in theoligonucleotide patterns obtained after ribonu-clease digestion of 16S and 23S rRNA when therewas a change in the growth conditions of thebacteria from which they were isolated. Second,analysis of the terminal nucleotide sequence bothat the 5' terminus (314, 321) and the 3' terminus(190) showed more than one terminal sequence.Finally, Young (359) fractionated rRNA byusing column chromatography and found atleast two separate peaks, both of which containedboth 16S and 23S rRNA. Small differences inoligonucleotide patterns from these two fractionsobtained after ribonuclease digestions were re-garded to be significant. Similar observationswere made with other microorganisms (194).Most recently, both 23S rRNA and 16S rRNAhave been separated into several components byusing density-gradient polyacrylamide gels (277)or agarose-acrylamide gel electrophoresis (57).However, none of these experiments proves theexistence of rRNA heterogeneity. Terminalsequence data obtained by Takanami (321)show that one unique sequence is always domi-nant, suggesting a considerable homogeneity.Presence of other "terminal" oligonucleotidesat the 5' terminus or at the 3' terminus may bedue to degradation of RNA during preparationor terminal analyses, or both, or to metabolicinstability. The metabolic instability of the 3'-endof tRNA causing apparent heterogeneity of the3' terminal sequence of isolated tRNAs is awell-known fact. Appearance of more than onepeak of rRNA on column chromatography oron acrylamide gel electrophoresis can be ex-plained on the basis of conformational differ-ences. Young's own data show that the relativeamounts of rRNA in his two fractions are alteredby heating and other conditions which are knownto alter the conformation of rRNA.

Furthermore, the sequence analysis of meth-ylated oligonucleotides of rRNA by Fellner andSanger (76), as described above, shows that manylong oligonucleotides with unique base sequencesand a chain length up to 11 occur in 1, 2, or 4moles per mole of RNA, and never in an amountless than 1 mole. Thus, both 16S and 23S rRNAfrom E. coli are largely homogeneous, at leastwith respect to the base sequence around methyl-

ated nucleotides. Further extensive analysis offragments of 16S rRNA performed by Fellneret al. strongly indicated homogeneity of 16SrRNA (74, 75). Completion of such base se-quence analyses will undoubtedly give a moreconvincing answer to the question of rRNAheterogeneity. It should be noted that the basesequence of 5SRNA from E. coli has proved thehomogeneity of this RNA species (31), althoughthere is a multiplicity of cistrons for this RNA inB. subtilis genome (287).

Base sequence analysis of rRNA. We havealready discussed some fragmentary data avail-able in connection with chain length, subunits,sequence homology, and heterogeneity of rRNA.It is clear that complete sequence analysis ofrRNA is important for our comprehensiveunderstanding of the structure and function ofrRNA. However, 16S rRNA alone containsabout 1,700 nucleotides, and it appears a for-midable task to sequence it completely. Yetmany recent technical improvements, especiallythe development of a new two-dimensionalelectrophoretic separation of oligonucleotidesdescribed by Sanger and his co-workers (271),and several new techniques for fractionatinglarger oligonucleotides (30, 62) have broughtthis goal closer. These methods have been suc-cessfully applied in the elucidation of the basesequence of 5S RNA, several tRNAs, and largefragments obtained from viral RNA. A seriousattempt to do a complete sequence analysis ofrRNA is currently under way (74, 75).

Secondary Structure of rRNA in Isolated Statesand in the Ribosome

The secondary structure of isolated rRNA, aswell as of the RNA in the ribosome in situ, wasfirst studied by Spirin's and by Doty's groups.Earlier studies are summarized by Spirin (295).Further studies on the secondary structure ofrRNA have been essentially an extension andelaboration of the earlier model that rRNAcontains many regions in which the single chaindoubles back upon itself forming hairpin-loopdouble-stranded helices connected by flexiblesingle-stranded regions (63).

It is generally believed that the secondarystructure of rRNA is the same before and afterthe removal of ribosomal protein by phenolextraction. This is mostly based on the followingobservations. (i) X-ray powder or wet gel diffrac-tion patterns of ribosomes and isolated rRNAare similar (145, 363). (ii) Degrees of hyper-chromicity obtained by heating ribosomes andisolated rRNA are about the same. The latterconclusion was reached with yeast ribosomes

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(45) as well as with E. coli ribosomes (193, 280,322). (iii) Optical rotatory dispersion studiesalso showed that the amount of base pairing inE. coli ribosomes and isolated rRNA in solu-tions of appropriate salt concentrations is aboutthe same (60 to 70%) (35, 191, 274). It should bepointed out, however, that most of these experi-ments simply show that the amount of secondarystructure is approximately the same both inrRNA in the ribosomes and in the isolatedrRNA. They do not demonstrate that the helicalregions are at the same positions. Furthermore,no definitive conclusions can be made about thetertiary structure of rRNA in intact ribosomesrelative to that of isolated rRNA.The amount of base pairing in the isolated

rRNA was measured by physicochemical meth-ods. Most of the measurements have given aboutthe same value: 60 to 70% of the total nucleotideresidues are involved in base pairing. However,there was less agreement on the composition ofthese base pairs (35, 48). Recently, Cotter andGratzer (44) applied infrared spectroscopy todetermine the extent and composition of basepairing. This method is based on the fact thatlarge shifts in the infrared absorption spectraassociated with the heterocyclic rings of nu-cleotides take place when base-paired poly-nucleotide structures are formed (131, 197, 198).This method appears to be more reliable thanother physicochemical methods since it is rela-tively insensitive to the base stacking of single-stranded chains, to the length of double helices,and to base sequences. With this method, theamount of AU base pairing was found to be27 to 29% and that of GC pairing 33 to 35%of the total nucleotide residues, respectively,for both E. coii rRNA and yeast rRNA (44, 325).The extent and composition of base pairs in

RNAs can also be determined by chemicalmethods by using specific reagents which reactwith certain base residues when these residuesare unpaired. Cramer and Erdmann (50) esti-mated the number of adenine residues whichare not base paired in E. coil rRNA by usingthe monoperphthalic acid oxidation method.They found about 20% AU base pairing bothin 16S and 23S E. coli rRNA. Thus, the valuethey obtained does not disagree to any largeextent with that obtained with the infraredspectroscopic method.A more-detailed structure of the helical regions

of rRNA has been obtained by Spencer andhis co-workers using "crystallizable" fragmentsof yeast rRNA. These fragments are obtainedby the mild alkaline degradation or enzymaticdigestion of yeast rRNA to a size corresponding

to a molecular weight of 9,000 to 20,000 (292,293). X-ray diffraction studies of these rRNAfragments showed that they contain helicalstructures very similar to those in the A form ofDNA, that is, double-stranded helical structureswith Watson-Crick base pairing (86). Althoughit is still not established that the secondarystructures present in the degraded fragmentsare identical to those in the intact rRNA, it isvery likely that this is so. The average size ofhairpin loops with helical structure has beenestimated to be about 25 nucleotides for therRNA of the smaller ribosomal subunit and 35for the rRNA of the larger ribosomal subunit ofrabbit reticulocytes (49).

RNA-Protein Interaction and the InternalOrganizaton of Ribosomes

It is possible to assemble 30S particles fromfree 16S rRNA and a mixture of about 20different ribosomal protein molecules (331).It was found that the assembly reaction requiresthe presence of specific rRNA. In the absence ofrRNA, no particles resembling 30S ribosomalsubunits were formed. Furthermore, neither17S cytoplasmic rRNA from yeast nor "16S"RNA prepared from E. coli 23S rRNA couldreplace the 16S E. coil rRNA in the reconstitu-tion. With these two RNAs, no particle sedi-menting at 30S was formed. Most of the RNAwhich interacted with protein formed insolubleaggregates. Small amounts of soluble particlesrecovered from the reconstitution mixtureswere very heterogeneous and lacked any func-tional activity in cell-free protein-synthesizingsystems. The molecular weight of yeast cyto-plasmic 17S rRNA is 6.5 x 105 (33) and isclose to the molecular weight value (5.5 X 106)for E. coli 16S rRNA. The "16S" RNA from 23SrRNA was prepared according to the methoddescribed by Midgley (196), and presumablyhas a size similar to that of 16S rRNA (196).Rat liver 18S rRNA also could not replace 16SE. coli rRNA in the reconstitution. These ex-periments clearly show that the rRNA-ribosomalprotein interaction is specific and is importantfor the over-all organization of ribosomalparticles.

Several questions may be asked about thespecific RNA-protein interaction in relationto the internal organization of the ribosome.(i) Are all of the ribosomal proteins involved inthe RNA-protein interaction or are there someribosomal proteins which assemble into theribosomal structure only through protein-proteininteraction? (ii) Are the ribosomal proteinsbound to the helical region of the rRNA or to

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its single-stranded region, or to both? (iii)What is the nature of the RNA-protein inter-action and what is the basis for its specificity?(iv) Does the RNA-protein interaction induce aconformational change of the rRNA; that is,is the tertiary structure of the isolated RNA thesame as that in the ribosome?Most of the models proposed for ribosomal

structure assume that all the ribosomal proteinshave specific binding sites on the rRNA (45,48a, 204, 295). The major reason given for thisassumption is that both 50S and 30S ribosomalsubunits can be "unfolded" without losing theirproteins. The unfolding of ribosomes refers tothe conformational changes that ribosomalparticles undergo upon removal of Mg+2 ion.They change from compact to less-compactstructures with lower sedimentation coefficients.This phenomenon has been studied by a numberof workers (35a, 92, 93, 193, 224, 295, 298, 349,352). Upon removal of Mg+2, the 50S ribosomalsubunit changes, via at least two discrete inter-mediates, to a particle sedimenting at about 19S.Similarly, the 30S ribosomal subunit changes toan unfolded particle sedimenting at about 16S.Unfolded ribosomal particles have a higherintrinsic viscosity and an increased susceptibilityto ribonuclease. It has been concluded thatthe observed changes reflect destruction ofthe compactly folded tertiary structure dueto electrostatic repulsion. The latter resultsfrom the removal of Mg+2 ions which nor-mally neutralize the RNA phosphates in theribosomes. All the ribosomal proteins arestill thought to be bound to the RNA chainin the unfolded state.Most recently, Moller and co-workers (205)

presented an argument which supports the beliefthat each ribosomal protein has its own bindingsite on the rRNA chain. They treated unfoldedE. coli ribosomes with nonspecific nucleasessuch as ribonuclease I from E. coli. It was foundthat about 15% of the total RNA is protectedfrom hydrolysis due to the binding of proteins.This protein-protected RNA is precipitatedduring the ribonuclease treatment. About 70to 80% of the total ribosomal protein was in-volved in the RNA protection and was pre-cipitated. The protected RNA fragments isolatedfrom the precipitate had an average chain lengthof about 30 nucleotide residues. From this itwas calculated that the molar ratio of protectedRNA to precipitated proteins is about one,supporting the concept of one binding site foreach of all the proteins. However, the calculationis only approximate. Moreover, as Moller et al.admit, it is possible that the RNA-protein

binding sites found in the precipitated aggregatesare produced during unfolding, nuclease diges-tion, or precipitation, and are not present inthe ribosome in situ. Other observations alsocast some doubt on the assumption that theposition of proteins on the rRNA in unfoldedparticles represents that in intact ribosomes.Traub and Nomura (332) succeeded in "re-folding" the unfolded 30S ribosomal subunits("16S" particles) back to compact 305 particleswhich are functionally active. The conditionsrequired for this refolding process were foundto be very similar to those found for the recon-stitution of 30S ribosomal subunits from 16SrRNA and proteins: moderately high ionicstrength (about 0.4) and heating (about 10 minheating at 40 C). It was found that about halfof the ribosomal proteins dissociate from theunfolded 30S ribosomal subunits under theseconditions. A decrease in salt concentration toprevent this dissociation prevents the refoldingprocess. On the other hand, it is likely that thepositions of the proteins which were not dissoci-ated in the above experiments are the same bothin the unfolded ribosomes and in the intactribosomes. More critical experiments are neces-sary to determine whether all the ribosomalproteins are involved in direct binding to rRNA.

Recent experiments in our laboratory as wellas in Kurland's laboratory suggested that atleast 6 out of 21 30S ribosomal proteins havespecific binding sites directly on the 16S RNA(201; Kurland, personal communication. Detailsof the experiments will be described later).The next question we shall consider is whether

the ribosomal proteins are bound to the helicalregions or to the single-stranded regions ofrRNA. Cotter, McPhie, and Gratzer (45) pro-posed that the ribosomal proteins are bound tothe single-stranded regions of rRNA and not tothe helical regions. They observed a close coin-cidence of the melting curves of isolated rRNAand ribosomes from yeast. Since the binding ofbasic proteins to DNA is known to stabilizethe double helical structure and to raise themelting temperature (Tm) at moderate ionicstrengths (16, 187, 341, 345), the observedabsence of stabilizing effects of ribosomal pro-teins was interpreted to mean that they are notbound to the helical regions of the RNA. Thissuggestion seems reasonable, but the experimentspresented do not prove it. Due to the presence oflatent ribonucleases (69, 280), it was difficultto distinguish the hyperchromic effect due tomelting of secondary structure from that dueto the hydrolysis of RNA in the ribosomes. Only

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recently, with the use of mutants of E. coilwhich are deficient in the ribosome-boundribonuclease I, could these difficulties be mini-mized. Using the ribosomes from such an E. colimutant strain, Tal (322) and Miall and Walker(193) were able to show some definite stabilizingeffects of ribosomal proteins on rRNA helicalstructures; the melting curve was much sharperand the Tm was higher with the ribosomes thanwith the isolated rRNA. Miall and Walkernoted that the unfolded 50S ribosomal subunits[in 10-2 M ethylenediaminetetraacetate (EDTA)]had the same total amount of secondary struc-ture as the compact 50S ribosomal subunits,and that the unfolded ribosomal particles showedthe same melting profile as the isolated 23SrRNA under the same conditions (in 10- MEDTA). Thus, in contrast to their stabilizingeffect on the helical regions of the native ribo-some, the ribosomal proteins present in the un-folded particles do not appear to stabilize thedouble helical structure of rRNA.

In any case, the stabilization of double helicalstructure in the ribosome does not necessarilyprove the direct interaction- between proteinsand double helical regions. A more direct ex-perimental approach was initiated in our labora-tory by the use of an in vitro ribosome recon-stitution system. RNA (16S) was treated withreagents, such as monoperphthalic acid (50, 51)or water-soluble carbodfimide (15, 97), thatreact only with bases in single-stranded regions.It was found that the treated 16S rRNAs wereinactive in reconstitution. Alteration of onlya few (less than four) adenine residues per 16SrRNA molecule to the N-oxide form by mono-perphthalate abolishes the reconstitution activityof the RNA (Held and Nomura, unpublisheddata). The inactive RNA was shown to be physi-cally intact. However, it failed to pick up severalribosomal proteins and it formed particles whichsedimented more slowly than 30S. Water-soluble carbodiimide also abolished the abilityof 16S rRNA to assemble into 30S ribosomalparticles; instead, protein-deficient inactive par-ticles were again produced (Mansour andNomura, unpublished data). These experimentsstrongly suggest that single-stranded regions areimportant in RNA-protein interaction, but donot exclude the possible additional involvementof helical regions in the interaction.We have very little experimental information

concerning the chemical nature of the specificRNA-protein interactions involved in ribosomeassembly. To explain the specificity demon-strated in the protein-RNA interaction, non-electrostatic force may have to be invoked.

However, the interaction must also involveelectrostatic force. It is known that nearly allthe ribosomal proteins can be split off from theRNA by various kinds of salts at high con-centrations if the ribosome is first unfolded byremoval of Mg+' (12, 133, 192, 301) or if thecompact ribosomal structure is first disruptedby urea (300). Since urea is known to destroynonionic bonds such as hydrophobic bonds(324, 353), these bonds may play an importantrole in maintaining a compact ribosomal struc-ture, through protein-protein or RNA-proteininteractions.The chemical basis of the specificity demon-

strated in the RNA-protein interaction is still amatter of speculation. It is conceivable that (i)a stretch of nucleotides with a specific basesequence is recognized by a specific structureof ribosomal proteins, (ii) a conformation of aregion of rRNA rather than a particular sequenceassures the "fit" of only a particular ribosomalprotein with a suitable conformation, or (iii)both of the above. It is conceivable that severalhydrogen bonds or hydrophobic bonds, orboth, may be involved in such specific recogni-tion in addition to the general electrostaticforces that ensure the over-all stability of thespecific interaction. In addition, the possibilityexists that only a few crucial proteins select thespecific region and make a "nucleus" and thatthe specificity of all the other interactions con-tributing to the assembly are of the protein-protein type. Indeed, this appears to be the casefor 30S ribosomal subunits.There are several studies on model systems

that indicate some selectivity in the interactionbetween basic proteins and nucleic acids. Forexample, polylysine appears to have a higheraffinity for the regions of RNA that are richin guanine and cytosine than for those rich inadenine and uridine (289). However, we haveno idea how relevant the gross selectivity studiedin these model systems is to the high specificityof the RNA-protein interaction in the ribosomalstructure.The last topic we shall consider is the tertiary

structure of rRNA in the ribosome. Althoughthere are several electron microscopic studiesof ribosomal particles (34, 114, 116, 132, 172,223), it is still not possible to obtain any detailedinformation concerning their internal structure.X-ray diffraction studies of intact ribosomes(145, 161, 363) do not give much informationdue to the lack of techniques for crystallizingribosomes and to the complexity of their struc-ture. However, we can be certain that the tertiarystructure of rRNA in ribosomes is different

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from that of isolated RNA. For example, theeffective hydrodynamic volumes of isolated 23Sand 16S rRNAs are much larger than those ofthe 50S and 305 ribosomal subunits, respectively,even under conditions in which the RNAs showtheir maximal degree of folding (193). It appearsthat the RNA per se is not able to take on thecompact conformation that exists in the ribosomein situ without the help of ribosomal proteins.Thus, protein-RNA or protein-protein interac-tions appear to induce conformational changesof rRNA. As we shall discuss in a later section,the assembly of 3QS particles from 16S rRNAand about 20 different protein molecules is ahighly cooperative process. Such cooperativeassembly might reflect an orderly conformationalchange of the rRNA during the assembly process.From the foregoing discussion, it is clear that

we cannot make any detailed model of ribosomestructure. The topological relationship among themany different molecular components is un-known. Even the very elementary questions asto which proteins are on the surface and whichproteins are buried inside, or which part of therRNA molecule is exposed on the surface ofthe ribosome, are not known. However, webelieve that at least a part of the rRNA is ex-posed and thus the ribosomal structure is drasti-cally different from the common spherical virusstructure in which the RNA is completely pro-tected by an outer protein shell. For example,the dye binding experiments by Furano, Bradley,and Childers (88) and the ability of ribonucleaseto reach rRNA suggest the surface location ofa part of the rRNA (77, 273, 285).Although we may have to use X-ray or elec-

tron microscopic methods for the eventual com-plete elucidation of ribosomal structure, so farthese techniques have provided little informa-tion. The most hopeful approach for the presentis perhaps a chemical one. For example, treat-ment of ribosomes with some specific bifunc-tional reagents may reveal neighborhood re-lationships between protein components. Analy-sis of the sequence of events in the assembly of30S ribosomal subunits in vitro may also givesome information about the topological relation-ships among protein components.

FUNCTIONAL ANALYSIS OFMOLECULAR COMPONENTS

Two possible roles can be considered for thecomponents of the ribosome. (i) A given com-ponent may be essential for the assembly processbut not required for any ribosomal function inthe finished ribosome. (ii) A given componentmay be required for some ribosomal function.

Several different approaches have been takento attack the problem. The first approach is thechemical modification of ribosomes to correlatethe resultant functional alterations with thechemical alterations of the component moleculesThe second approach is genetic. The identifica-tion of altered components and the correspond-ing altered functions in mutant ribosomes maygive information as to the role of these com-ponents in normal ribosomes. A third approachis the use of reconstitution techniques such asthose developed in our laboratory. This lastapproach is the most useful for the systematicanalysis of many ribosomal components simul-taneously, and it is especially powerful in com-bination with the first two. We shall first de-scribe that of using the reconstitution technique.

Reconstitution of RibosomesReconstitution of ribosomes is the most

direct approach to the study of the functionalrole of ribosomal components, and also to themechanism of assembly. However, no seriousattempt at reconstituting ribosomes was madeuntil a chance observation induced attempts atpartial reconstitution from protein-deprivedribosomal particles and the removed proteins.The complexity of ribosomes may have madepeople pessimistic about the success of the ven-ture. It was also quite possible that the assemblyof ribosomes might involve several nonribosomalcomponents as templates or enzymes, and thispossibility may also have discouraged people.In fact, it was only the successful demonstrationof the reconstitution of functionally active30S particles (331) that established that all in-formation for the correct assembly of the 30Sribosomal subunit is contained in the structureof its molecular components and not in anynonribosomal factors.

Partial reconstitution. In 1961, Brenner, Jacob,and Meselson (25) noted the presence of tworibonucleoprotein peaks when they centrifugedbacterial extracts containing ribosomes in aCsCl density gradient. The lighter band (theB band), corresponding to a density of 1.61,contained mRNA as well as proteins beingsynthesized; the heavier band (the A band),corresponding to a density of 1.65, did not.Later study (192) revealed that the B band con-tained undegraded 50S and 30S subunits,whereas the A band consisted of a mixture ofsmaller 40S and 23S "core" particles. The latterare created from the 50S and 30S ribosomalsubunits, respectively, by the splitting off ofabout 30 to 40% of the protein during the den-sity-gradient centrifugation. The split protein

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was found at the top of the gradient. Neitherpurified 50S nor purified 30S gave the B band.The explanation for the presence of the B bandwhen crude bacterial extracts are used is thatsome of the ribesomes (70S particles in poly-somes) are resistant to splitting, perhaps be-cause they are stabilized by mRNA and growingpolypeptide chains.

It was then asked whether the 40S and 23Score particles are functional and whether thesplitting of the proteins is reversible. The ques-tions were studied both in Meselson's laboratory(305) and in our own (129). It was demonstratedthat both 40S and 23S core particles are inactivein in vitro protein synthesis and that the splittingoff of proteins is reversible; functionally active50S particles can be reconstituted from 405cores and the split proteins from 50S subunits,and active 30S particles can be reconstitutedfrom 23S cores and the split proteins from 305subunits. This reconstitution reaction is spon-taneous and rapid; it is complete within a fewminutes at 37 C (235). Split proteins were shownto be distinct from core proteins by gel electro-phoresis (94, 334) as well as by functional tests(334). The split proteins were fractionated andthe functional role of each of the separatedfractions was studied (330). For example, splitproteins from 50S subunits were separated intotwo fractions, an acidic fraction (SP50A) and abasic fraction (SP5OB). Without SP5OA, theparticles formed were completely inactive inpoly U-directed polyphenylalanine synthesis.However, the particles prepared from 40S coresand the SP5OA fraction in the absence of SP50Bshowed partial activity. It was concluded thatsome proteins contained in the SP5OA fractionare essential for 50S function, but that proteinsin the SP5OB fraction are only stimulatory.Similarly, the split proteins from 30S particleswere separated into two fractions, SP30B andSP30A. The SP30B fraction was essential forall the functional tests examined, whereas theSP30A fraction was shown to be only stimula-tory. The four protein fractions, SP5OA, SP5OB,SP30A, and SP30B, could not be interchangedin functional analyses. The SP30B fraction wassubsequently separated into its five individualcomponent proteins, and their functions werestudied; three of the proteins were shown to beessential for both mRNA-directed tRNA bindingand poly U-directed polyphenylalanine synthesis,whereas the two other proteins were shown to bestimulatory (329).

These experiments established the functionalheterogeneity of ribosomal proteins and werelater used to support the finding of the chemical

heterogeneity of ribosomal proteins as revealedby gel electrophoresis. The demonstration of theparticipation of many proteins in one function(e.g., poly U-dependent phe-tRNA binding)suggested a highly organized ribosomal struc-ture. The experimental results posed severalquestions. For example, some proteins wereshown to be stimulatory but not essential, andthe possible functions of such stimulatory pro-teins were considered. This question was studiedwith respect to proteins in the SP30A fraction(335). Binding experiments in the absence of50S subunits, with either poly U-directed phe-tRNA or f2RNA-directed fMet-tRNA bindingsystems, indicated that the stimulation by SP30Aproteins is due to an increase in the number ofspecific binding sites and, therefore, probablyin the number of active particles in the prepara-tion. It was suggested that these "stimulatory"proteins are not directly involved in the pertinentfunction and that in the absence of these pro-teins the ribosomal particles can take severalstates, some active and others inactive. Thepresence of the stimulatory proteins permitsonly the active state. Recently, Kurland (154)suggested another possible explanation: thatribosomal populations are heterogeneous, andthat the stimulatory proteins are required forthe function of some ribosomes but not for thefunction of others which are already active inthe absence of these pertinent stimulatory pro-teins.The definitions of split proteins and core

particles are operational. In the original experi-ments, centrifugation in 5 M CsCl containing0.04 M MgCl2 was used to obtain the split pro-tein and core fractions. It was shown that thekinds of proteins split off from the ribosomeparticles by salt treatments vary depending onthe kinds and concentrations of salts used andthe concentrations of stabilizing divalent cationsas well as the conditions of centrifugation (12,133, 166, 185, 261, 301, 304). Thus, various kindsof "core" particles were obtained and the re-constitution of functional ribosomal particlesfrom these various particles and the correspond-ing split proteins was demonstrated (13, 304).Although the complete reconstitution system for30S subunits has somewhat diminished interestin these partial reconstitution systems, the func-tional analysis of 50S ribosomal componentsis still dependent on these systems since totalreconstitution of 50S subunits has not beenachieved. Staehelin, Maglott, and Monroe (304)used a method which causes a stepwise re-moval of some of the 50S proteins yielding a-,13- and 'y-core particles, in order. Peptidyl trans-

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ferase activity assayed by the method of Monroand Marcker (211) was found in a- and (3-coreparticles, but not in y-core particles. The (3 coreis deficient in five to six proteins, (SP50od pro-teins), and the y core is missing an additionalfive proteins (SPY z proteins). The peptidyltransferase activity could be restored by mixingthe y-core particles and the SP#-y proteins.Thus, one or more proteins in the SP#-. fractionmust play an important role in the catalyticactivity of 50S subunits. However, the splitproteins by themselves did not show any peptidyltransferase activity, implying that the peptidyltransferase reaction is catalyzed by a structureconsisting of more than one 50S protein or by a50S protein whose active conformation is ob-tained only in the presence of other 50S proteins.

It is interesting to note that the removal ofone group of proteins (SPO a) not only abolishespeptide bond formation but also abolishes thebinding of chloramphenicol. Thus, it appearsthat chloramphenicol inhibits peptide bondformation by interacting directly with the struc-ture responsible for peptidyl transferase ac-tivity. Another interesting observation is thatalthough the # core is active in the peptidyltransferase reaction, its activity in poly U-di-rected polyphenylalanine synthesis is weak.Thus, some proteins in the SP50 protein fractionappear to be essential for other 50S functions,although they are not required for the peptidyltransferase activity.

Complete reconstitution. Complete reconstitu-tion of 30S ribosomal subunits from free RNAand proteins was achieved in our laboratory(331). The 16S RNA used was obtained from30S subunits (or 23S core particles) by phenoltreatment, and a mixture of 30S proteins wasprepared using the urea-LiCl method of Spitnik-Elson (300). Two factors were important forthe success of reconstitution. One was the heat-ing of the reconstitution mixture (20 min at37 to 40C), and the other was the use of a ratherhigh ionic strength of 0.37 (332).The complete reconstitution system allowed

the study of the functional role of the 16S RNA.It was first shown that RNA must be presentfor the assembly reaction to occur, since in theabsence of 16S RNA no soluble particles re-sembling the ribosome are formed. Moreover,cytoplasmic 17S ribosomal RNA from yeast or18S ribosomal RNA from rat liver cannot re-place E. coli 16S RNA in the formation ofparticles sedimenting at 30S (331).However, some 16S RNAs from distantly re-

lated bacterial species, such as Azotobactervinelandii or B. stearothermophilus, can replace

E. coli 16S RNA and form functionally activehybrid 30S particles with E. coli 30S proteins(236). The reverse combinations, that is, 30Sribosomal proteins from A. vinelandii or B.stearothermophilus with 16S RNA from E. coli,also gave active hybrid 30S particles. Although16S RNAs from these three different bacterialspecies have some portions of their base se-quences in common, large portions are different.Thus, the requirement for a specific base se-quence in ribosomal RNA is not absolute. Itwas suggested that only certain small regions of16S RNA are directly involved in specific in-teraction with ribosomal proteins in the as-sembly of ribosomal particles and that theseregions represent "conserved" regions, that is,regions having base sequences which are commonor very similar among different bacterial species.These and other reconstitution experiments

have provided information on the role of 16SRNA in the assembly of ribosomal particles butnot on the possible role of this RNA in the trans-lation process.On the other hand, the reconstitution system

has already provided considerable informationon the functional role of each of the 30S ribo-somal proteins. First, the 30S protein mixtureused in the original experiments was replacedwith a mixture of 19 purified ribosomal proteins.It was shown that functionally active 30S par-ticles can be reconstituted from the purified 16SRNA and this mixture (234). The reconstitutionswere then performed with 16S RNA and a pro-tein mixture with one component omitted. Weasked whether physically intact 30S particleswere performed and, if so, whether they werefunctionally active. Sedimentation analysis ona sucrose gradient was used to judge the effective-ness of physical reconstitution, and variousknown 30S functions were analyzed to deter-mine the functional capability of the proteindeficient particles. Typically, the following as-says were used: poly U-directed phenylalanineincorporation, poly U-directed incorporation ofincorrect amino acids (a mixture of isoleucine,tyrosine, and serine) in the presence of strepto-mycin, phage f2 RNA-directed incorporation ofvaline, poly U-directed phe-tRNA binding, andAUG triplet-directed binding of fMet-tRNA.The last assay was done in two ways. In the firstmethod, fMet-tRNA binding was measured at0 C in the presence and absence of initiationfactor F2 and the amount of stimulation by F2was used as a measure of ribosome function.In the second, binding at 30 C in the presenceof F2, with and without F1, was measured todetermine stimulation by Fl. By comparing

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these results with those obtained for ribosomesreconstituted with the complete protein comple-ment, it was hoped that any ribosomal proteinswhich specifically interact with these initiationfactors could be identified.The results obtained so far in our laboratory

from such functional analyses are summarizedbelow (see also Tables 2 and 3).

(i) Individual omission of most of the pro-teins, except P1 and P38, caused a significantdecrease in some of the functional activities ofthe reconstituted particles. Both P1 and P3a aredefinite ribosome-associated proteins and havebeen detected and isolated by all the groupsworking on ribosomal proteins. Three possibleexplanations may be given for their behavior.(a) These proteins are genuine ribosomal pro-teins and are functional in vivo but have beeninactivated during purification in the presence ofurea. (b) These proteins have functions whichcannot be detected by the activity assays used.(c) These proteins are not genuine ribosomalproteins but are proteins tightly bound to the30S ribosomal particles, like ribonuclease I.This last possibility is more likely with proteinP1, since according to Kurland et al. (157)there is only 0.1 to 0.14 molecule of P1 per 30Sparticle. In any event, it is satisfying that mostof the purified proteins show effects in vitro andhence appear to represent genuine ribosomalcomponents.

(ii) Several proteins were clearly found to beimportant for the physical assembly of the ribo-some. Omission of these proteins producedparticles sedimenting at 20 to 25S rather thanat 30S (compare Table 2). As expected, theseparticles showed greatly reduced activity in allthe functional tests employed (except the mis-reading of poly U by [-P5] particles). The pro-teins in this category are P4a. P4b, P5, P8, and Pg.When the protein composition of [-P4b] par-ticles was analyzed, it was found that at leastthree proteins beside P4b were missing. Hence,the presence of P4b is essential for the bindingof these other proteins. It is likely that otherparticles are also deficient in several proteinsbeside the one omitted in each case. This in-ference is supported by the observation thatparticles obtained by omitting one of these pro-teins cannot be reactivated by subsequent ad-dition of the protein omitted (Mizushima andNomura, unpublished data). Thus, these proteinsappear to be required for the assembly of the30S subunit.

(iii) Omission of some other proteins (P3, P4,P6, P7, P11, P13) was found to produce particleswhich sediment slightly slower than 30S par-

ticles. These deficient particles were inactive insome or all of the functional tests. Several otherproteins (P2, Plo, Plowa P12, P14, and P15) areapparently not required for assembly as judgedby the sedimentation analysis, but are still re-quired for some or all 30S functions. Amongthese proteins, P12 and P14 were found also tobe required for the binding of some of the otherproteins, although their absence produced nor-mally sedimenting particles (201; see Fig. 3).One of the primary objectives of the recon-

stitution approach was to identify proteins es-sential for ribosome function by correlatingtheir omission with the loss or alteration of aribosomal function. To make a strong inferencefrom such a correlation, it must be shown thatthe effect of the omission is direct. Normal sedi-mentation behavior does not guarantee that allthe proteins initially added are present in thefinal particle, and it is possible that the omissionof a protein which is not essential per se couldprevent the binding of a protein which is. Torule out this possibility, it is necessary to showthat all the proteins except the one being omittedare present in the deficient particle. This can bedone in two ways: either by direct analysis ofthe protein composition or by showing that amissing function can be restored by addition ofthe missing protein to the isolated, deficientparticle under reconstitution conditions. Thesecond test was used in our preliminary studiesfor most of the proteins (234, 250; Mizushimaand Nomura, unpublished data). With the pro-teins required for assembly mentioned above(P4b, P5, P9, P8) as well as some others (e.g.,P6), restoration of activity was not observed.On the other hand, with some other proteins(e.g., Plo) restoration of activity was clearlydemonstrated upon addition of the pertinentprotein to the corresponding deficient particle.The latter result suggests that the effects ob-served upon omission of these proteins aredirect results of the absence of these proteinsin the particles. This does not necessarily meanthat these proteins constitute "active sites."It is very likely that many proteins are indi-rectly required for function, because they mustbe present to keep the active site proteins in anactive conformation.

(iv) Both Plo and P7 were found to be uniquewith respect to their effect on the frequency oftranslational errors. The properties of P0o-deficient particles ([-P10] particles) were studiedin detail (250). In the presence of 0.015 M orhigher Mg+' concentration, [-P1o] particles are70 to 90% as active as the control [E Pi] particlesin both amino acid incorporation and tRNA

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binding directed by synthetic mRNA. However,they are very weakly active in the initiation func-tion, as judged by f2 RNA-directed valine in-corporation, fMet-tRNA binding directed byAUG or f2 RNA, and formyl-methionyl-puro-mycin formation, all at 0.01 M Mg+2. The mostunique feature of [-P1o] particles is a reductionin the misreading of poly U under variousexperimental conditions (compare Table 2).The protein Plo is controlled by the str locus.It was shown that [-Plo] particles do not bindradioactive dihydrostreptomycin (250). Thus, adecrease in streptomycin-induced translationalerrors was to be expected. However, severalother error-inducing agents also showed lessmisreading: kanamycin A, neomycin B, paro-momycin, ethyl alcohol, or a high concentrationof Mg+2. Thus, although the primary target ofthese agents may not be Plo, a specific structureof 30S ribosomal particles which involves Plo isimportant for their ultimate effect.The converse situation was observed with

TABLE 2. Functional analysis of30Sribosomal proteins"

Protein omit-ted from re-constitutionmixture

P45P4b

P5P8P5

P6P7PiOP1lP1sP2P5P4PloaP12P14P15

piP3a

Sedimentationpattern of

reconstitutedparticles

30S20-25S20-255

20-25S20-25S20-25S

28-30S28-30S30S28-30S27-30S

30526-30528-30530530S30S30S

30S305

Polyphenylala- Relative degreenine synt esiz- of misreadinging activity of poly Ub

1002313

212919

122443727

47485077436048

9392

1.01.71.2

2.41.10.84

1.56.50.42.11.2

1.91.01.21.31.71.91.7

1.11.0

aData from Nomura et al. (1969).bDegree of misreading [total amount of iso-

leucine, serine, and tyrosine incorporated in thepresence of streptomycin)/(amount of phenyl-alanine incorporated in the absence of strepto-mycin) ] is compared to the control value.

respect to protein P7. The [-P7] particles showeda pronounced increase in the streptomycin-induced misreading of poly U (Table 2). Highmisreading was also observed in the presence ofother agents such as neomycin B, ethyl alcohol,and 0.02 M Mg+'. The decrease in misreading ofpoly U by [-P1o] particles and the increasedmisreading by [-P7] particles are clearly dueto the lack of P1o and P7, respectively, sinceaddition of these proteins to the respective de-ficient particles restored the "normal" errorfrequency. The omission of no other singleprotein affects the error frequency significantly;these two proteins are thus unique in influencingthe error frequency and they act in oppositeways. Since both proteins are also required for

TABLE 3. Summary of stoichiometry and functionalrequirements of30S ribosomal proteins

30SProtein

Pi (A,)P2 (As)Ps (Bi)PS5P4 (B2)P45P4bP6Ps (Bs)P7

Ps (B4)PgP's

P555Pu (BO)P12P13P14P16PabPsc

Kurland'scode

4a923

102a84

11

126 (and 7?)

15

1412b12a131615a

57

Stoi-chio-metricclass'

FF

u

uuM

uu

FMFFF

Require-ment forreconsti-tution ofactive30S

particlesb

++

(_

++(++C++)(++)C++)++(++)C++)C++)++

++++++

C4)(I)

Note

Spc sensitivity

K character

Fidelity oftranslation

Str sensitivity;ambiguity oftranslation

" Data from Kurland et al. (1969): U, unit protein; F,fractional protein; -, data not available.

b Data from Nomura et al. (1969). See also Table 2. Sym-bols: (+ +), strong requirement presumably because of re-quirement in the assembly reaction-particle reconstituted inthe absence of these proteins sedimented at 20-25S (see Table2); ++, strong requirement; +, partial requirement; -, norequirement demonstrated; Ct), weak effects were shown inour previous studies. However, these proteins were probablypresent in Pi preparation used in these studies. In view of theirinvolvement in the assembly sequence (see Fig. 3), these pro-teins may also be required for the reconstitution of func-tionally active 30S particles.

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some other ribosomal functions, they are normaland essential ribosomal components. Theseresults formally prove the concept originallydeveloped by Gorini that translational am-biguity is influenced by ribosome structure.Since P7 is essential for maintaining the fidelityof translation, it is probable that it participatesdirectly in the selection of the right tRNA,that is, in codon-anticodon interaction. As hasbeen pointed out by Kurland (154), the selectionstep may be different from the final bindingstep. That is, a weak specific interaction betweencodon and anticodon could be used to trigger astrong nonspecific interaction of tRNA with theribosomes, leading to the formation of a specific,stable complex.The function of P10 is more intriguing. Omis-

sion of Plo only slightly affected the extent ofspecific tRNA binding assayed with varioussynthetic mRNAs, but had a greater effect onthe binding of fMet-tRNA directed by AUGor f2 RNA (234, 250). It is possible that P10is not directly involved in the tRNA binding sitebut is involved only in the interaction with theinitiation factors. The ambiguity caused by thepresence of Plo may be the result of a directphysical interaction of P1o with P7, leading toinhibition of the function of P7 (maintenance offidelity), or the presence of Plo may somehowincrease ambiguity without any direct interactionwith P7. There is further discussion of this prob-lem in a later section.

(v) There are several other unique proteinsaffecting only some functions. As mentionedbefore, P1o is essential for the initiation function,especially that part stimulated by both initiationfactor F1 and F2, but not for other tRNAbinding. P14 appears to be similar to Plo in thisrespect but is functionally necessary to a lesserextent. P15 appears to be required for the functionof initiation factor F2 but not of Fl. Thus, theseproteins may constitute a part of the ribosomestructure with which the initiation factors inter-act. Since pure radioactive initiation factor F1can now be obtained and its physical interactionwith the 30S subunit is demonstrable (120), theabove conclusion can be directly confirmed.

It is evident that the reconstitution techniqueis useful and, coupled with other approaches,may eventually give much information on therole of all the molecular components of 30Ssubunits. A similar complete reconstitution sys-tem is not yet available for 50S subunits. Thepartial reconstitution system presently availableallows the functional analysis of only a part ofthe 50S proteins. Thus, the complete reconstitu-tion of 50S subunits is desirable.

Chemical Modification of Ribosomes andRibosomal Components

Chemical modification has been used exten-sively to study the relationship between thestructure and function of enzymes and, morerecently, of tRNAs.The approach has given especially useful in-

formation on the active sites of several enzymemolecules. This is due largely to the availabilityof many specific modifying reagents, the de-velopment of techniques that have made identi-fication of chemical alterations much easier, andstrategies such as substrate protection, site-specific labeling or affinity labeling. Thus, it wasnatural to apply similar approaches to the studyof ribosome structure and function.The first chemical modification experiments

were done by Moore (212). He treated ribosomeswith several group-specific reagents to identifythe chemical groups responsible for mRNAbinding, using binding of radioactive poly U toribosomes in the presence of 10-2 M Mg+2 toassess the mRNA binding function of ribosomes.Moore observed that formaldehyde, whichreacts with free amino and sulfhydryl groups inproteins as well as with free amino groups innucleic acids, causes a loss of poly U-bindingcapacity and of the capacity for poly U-directedpolyphenylalanine synthesis. Nitrous acid, whichreacts with free amino groups in RNA and withfree amino groups and several other functionalgroups in proteins, was also found to inactivatepoly U binding. Dinitrofluorobenzene, whichreacts with free amino groups in ribosomalproteins but not with those in RNA, did notinhibit poly U binding, although it abolishedpoly U-directed phe-tRNA binding and polyU-directed phenylalanine incorporation. Per-phthalic acid, which selectively oxidizes nucleo-tides and also reacts with several functionalgroups in proteins, inhibited poly U binding.From these results, it was concluded that RNAamino groups are vital for the mRNA bindingfunction of the ribosome.The interpretation of such experimental re-

sults is especially difficult because of the com-plexity of ribosomes. Except for dinitrofluoro-benzene, it was not determined whether thereagents actually reacted with RNA or withprotein. It is clearly important to identify thealtered components responsible for inactivationof the function being measured. If only a fewgroups per ribosomal particle are altered, itwould be easy to identify the altered componentschemically. In such a case, the use of specificradioactive or colored derivatizing reagentsmight be useful for the identification. The binding

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of a natural mRNA such as f2 RNA involvesspecific sites on the mRNA, the initiation factorproteins, and presumably fMet-tRNA. Thus,the relation between the capacity of ribosomesto bind poly U in the presence of a high con-centration of Mg+2 and the capacity to bindnatural mRNA is not clear. Moreover, poly Usometimes binds to ribosomes in nonspecificways. For example, the 23S core particles de-scribed in the previous section do not bind polyU, whereas particles reconstituted from 23S coresand split proteins from 30S subunits regain theability to bind poly U (256, 330). However,particles obtained by mixing 23S cores with splitproteins from 50S subunits at a low- or medium-salt concentration are also able to bind poly U(Nomura, unpublished data). Clearly, suchbinding is a nonspecific binding caused by anionic interaction between acidic poly U moleculesand basic proteins on the surface of ribosomalparticles. The only way at the moment to distin-guish nonspecifically from specifically bound polyU is to test the ability of the poly U-ribosomecomplexes to bind phe-tRNA. However, this testinvolves not only the mRNA binding ability butalso the tRNA binding ability of ribosomes.Although it is possible that the absence of poly

U binding after certain chemical treatments maybe meaningful, experimental data showing theoccurrence of poly U binding after certain chemi-cal treatments must be treated with caution. Thework done by Moore was, nonetheless, the firstsystematic approach using chemical modification,and it provided the data which served as thestarting point for further studies.Another useful general approach to the deter-

mination of function of ribosomal components isthe combined use of chemical and reconstitutiontechniques. For example, in preliminary experi-ments (Bowman and Nomura, unpublished datacited in 234), we have treated 30S subunits withnitrous acid and observed their inactivation in apoly U-directed polyphenylalanine-synthesizingsystem. It was found that 16S RNA extractedfrom the inactivated 30S subunits was active in areconstitution system in the presence of proteinsfrom normal 30S subunits. It was concluded thatthe inactivation of the 30S subunits by nitrousacid is due to inactivation of ribosomal protein.Although we have not identified the 30S proteinsresponsible for the inactivation, such identifica-tion is easy in principle if the reconstitutiontechnique is used.

This type of approach was also used by Cravenand his co-workers to study the structure-functionrelationships of 30S subunits (52). They usedtetranitromethane, which reacts with tyrosine

residues in proteins. It was found that treatmentof 30S subunits with tetranitromethane causedrapid inactivation of poly U-directed phe-tRNAbinding activity. The kinetics of inactivation fol-lowed the kinetics of nitration of tyrosine residuesclosely. That the loss of binding activity is actuallydue to inactivation of proteins and not RNA wasdemonstrated by the reconstitution technique.Significantly, it was observed that the ribosomescould be protected from inactivation by tetra-anitromethane by the presence of poly U plustRNA at 20 mM Mg+2. Thus, it appears that themodified proteins responsible for the inactiva-tion represent a part of the tRNA binding site.Although Craven et al. have not identified themodified proteins, their experiments indicate theusefulness of this type of approach for identifyingthe proteins directly involved in ribosome func-tions.

There are several other reports related to thechemical modification of ribosomes (91, 138). Weshall describe only the effects of several sulfhydrylblocking reagents. Tamaoki and Miyazawa (323)observed that sulfhydryl blocking reagents causedissociation of E. coli 70S particles into 30S and505 subunits, and that ,B-mercaptoethanol canprevent this dissociation at high temperatures(200). Traut and Haenni (338) observed thattreatment of E. coli ribosomes with N-ethyl-maleimide or dithiobis-(2-nitrobenzoic acid) re-sulted in the loss of 30 to 70% of their ability tosynthesize polyphenylalanine and that the inac-tivation was caused by reaction of the reagentswith the 30S subunits. The sulfhydryl reagentsdid not react with 50S subunits and thus did notinhibit the polypeptidyl transferase reactioncatalyzed by these subunits (210, 258, 339).Retsema and Conway (258) found that N-ethyl-maleimide inhiobits poly U-directed phe-tRNAbinding as well as the formation of 70S particlesfrom 50S and 30S subunits in the presence ofpoly U and tRNAs at 8 mm Mg+2, although itdoes not inhibit at 20 mm Mg+2. They suggestedthat sulfhydryl groups on the 30S subunits areinvolved in the association of 30S and 50S sub-units and, consequently, in the binding of sub-strates or polymerization factors to the ribosomalsurface.The experiments described so far have involved

the treatment of 70S particles or 50S or 30Ssubunits as a whole. Chemical modification canalso be applied to the separated individual mo-lecular components of ribosomes. Effects of thechemical modification of isolated 16S RNA onits reconstitution activity were investigated inour laboratory. It was found that E. coli 16S RNAis very sensitive to treatment with nitrous acid

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(236). The chemical analysis of base alterations(deamination of adenine, guanine, and cytosine)indicated that only a few (six to eight) base alter-ations are sufficient to destroy completely thereconstitution activity of one 165 RNA molecule.The inactive particles reconstituted with nitrousacid-treated RNA sedimented more slowly thannormal 305 particles. These and other resultsshow that nitrous acid destroys the ability of theRNA to be folded into its normal compact formin the ribosomal particle. Since the inactivationcan be explained merely on the basis of defectivephysical assembly, the nitrous acid experimentprovides no direct information about RNA func-tion in the finished ribosome. The effects ofmonoperphthalic acid on the activity of 16S RNAwere also investigated (Held and Nomura, un-published data). The treated rRNA was found tolose its ability to assemble into the normal 30Sparticle. Since monoperphthalic acid reacts withadenine and cytosine in single-stranded regions(50, 51), this suggests the importance of single-stranded regions of RNA in the assembly process.A similar conclusion was reached from experi-ments with water-soluble carbodfimide (Mansourand Nomura, unpublished data).Experiments can also be done using purified

protein molecules. Since we now know which 30Sproteins are directly involved in the assembly andwhich proteins are involved in ribosomal func-tions unrelated to assembly, we can select suitableproteins for study. The treatment of a proteinwith modifying reagents could give informationon the role of certain functional groups of theprotein in the assembly process or in the func-tion of the intact ribosomal particle.

Genetics of RibosomesTwo types of ribosome genetic studies can be

considered. One involves the isolation of mutantswhich have defects in the assembly or regulationof assembly of ribosomes. This gives useful in-formation on the mechanism of biosynthesis ofribosomes, and will be discussed in a later section.The second type of genetic study involves isola-tion of mutants with ribosomes having alteredproperties. Mutations in this class may involvethe genes for ribosomal proteins, for ribosomalRNA, or for nonribosomal components (e.g.,methylases) which can cause alterations in ribo-somal RNA or proteins. Since the ribosomesconsist of many protein molecules and RNA,many genes should be involved in ribosome func-tion and such a genetic study would be expectedto be a productive approach to the structure-function problem of ribosomes. In addition, themappipg of genes for these mutations would give

some information about the genetic control ofthe biosynthesis of ribosome components andabout the ribosome assembly process. However,there are only a few systems that have beenstudied in detail and which have given any usefulinformation. We shall limit our discussion mostlyto these systems. More extensive reviews of sub-jects related to ribosome genetics have beenpublished recently by Schlessinger and Apirion(281), and by Weisblum and Davies (348).Mutations at the str and ram loci. One of the

most useful techniques for isolating ribosomalmutants is to select for mutants resistant to anti-biotics that affect protein synthesis. Thus, strepto-mycin, spectinomycin, neomycin, kanamycin,kasugamycin, lincomycin, and erythromycinhave been used for this purpose. Streptomycin(Sm) has been studied the most extensively. Smkills wild-type E. coli cells. Among the survivorsin cultures treated with Sm, one can find twoclasses of mutants. One is resistant to Sm (Str-R),and the other is resistant to Sm, but in addition,requires Sm for growth. The latter is called Sm-dependent (Str-D). Spotts and Stanier (303) firstsuggested that the site of action of Sm is the ri-bosome and that the Str-R and Str-D mutations in-volve an alteration ofribosomes. Subsequent in vi-tro experiments confirmed this suggestion. It wasshown that poly U-directed polyphenylalaninesynthesis in a cell-free system with ribosomesfrom Str-S bacteria was inhibited by Sm, but thatthe same reaction with ribosomes from Str-Rbacteria or Str-D bacteria was not inhibited (78,294). Under certain conditions, Sm stimulatedin vitro polypeptide synthesis by ribosomes fromStr-D bacteria (170). In addition, the ribosomesfrom Str-S cells were shown to bind radioactivedihydrostreptomycin in vitro, whereas the ribo-somes from Str-R cells did not (139). The ribo-somes from Str-R cells are also resistant to Sm-induced misreading of synthetic mRNA (60). Thealteration responsible for the Str-R phenotypewas subsequently localized in the 30S subunit(47, 59): when 30S subunits from Str-R bacteriawere used in vitro, polypeptide synthesis wasresistant to Sm regardless of the origin of the 50Ssubunits. After the 30S subunit reconstitutionsystem was developed, it became possible toidentify the altered component in the Str-R 305subunits. It was shown that the sensitivity of thereconstituted 30S subunits depends on the originof the proteins but not of the RNA. Particlesreconstituted from proteins of Str-R cells andRNA were resistant to Sm whether the RNAused was from Str-R cells or not (333). The finalidentification of the altered protein was done inour laboratory (250). The 30S ribosomal proteins

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from both sensitive and resistant bacteria werefractionated, and suitable combinations of theproteins were used for the reconstitution. Thereconstituted particles were then tested for theirsensitivity to Sm in in vitro polypeptide synthe-sizing systems as well as for their ability to bindradioactive dihydrostreptomycin. In this way, itwas established that a single protein, which wehad designated Plo, determines the sensitivity ofthe entire 30S ribosomal particle to the inhibitoryaction of Sm, its sensitivity to Sm-induced mis-reading, and its ability to bind the antibiotic. Thealtered component in ribosomes from a Str-Dmutant was also investigated. The alteration wasfound to be localized in the 30S ribosomal sub-unit (170). With the reconstitution technique, itwas shown that the altered protein is identical tothat altered by the mutation to Str-R, namely,Plo (19). This conclusion is consistent with thefact that Str-D mutations map very closely toStr-R mutations (117), and are probably allelic(176). As will be described below, there are

several Str-R mutations all mapping at the strlocus but which are different from each otherwith respect to the restriction of genetic as wellas phenotypic suppression (103, 104). The proteinP1o can have several different structures dependingon the mutational alteration. This presumablygives rise to several different resistant phenotypesas well as to the dependent phenotype. Therefore,the str gene is, in all likelihood, the structuralgene for the protein P10.

Gorini and Kataja (105) discovered that certainmutational defects can be suppressed by Sm. Theyobserved that certain arginine-requiring strainscan grow without arginine if Sm is present in themedium. Their interpretation was that Sm inducestranslational errors by acting on ribosomes.Induction of translational errors by Sm was, infact, demonstrated in vitro. Davies, Gilbert, andGorini (60) showed that in in vitro polypeptidesynthesizing systems, Sm induced extensive in-corporation of amino acids that were not codedby the synthetic messenger RNA present. Suchmisreading was low when they used ribosomesfrom Str-R cells. From these experiments, theyproposed that ribosomes play an important rolein influencing the fidelity of translation. Sm was

thought to induce translational errors by inter-acting with a ribosomal component and somehowdistorting the ribosomal structure.Many Str-R and Str-D mutants were isolated

and their phenotypes were studied. One majorfeature of Str-R or Str-D mutations is theirpleiotropic effect. For example, Str-R mutants

which were selected by resistance to the killingaction of Sm showed the following properties.

(i) Restriction of the genetic suppression of non-sense mutations. Several workers found that some(but not all) mutations to Str-R cause restrictionof any genetic suppression ability which waspresent in Str-S parent strains; that is, these mu-tations inhibit the translation of nonsense codonsby suppressor tRNA in vivo (46, 90, 104, 159,249, 275). Several different Str-R mutations wereobtained showing different degrees of restriction.(ii) Restriction of phenotypic suppression by Sm.Gorini and his co-workers discovered that a veryefficient phenotypic suppression of an argF ambermutation is induced by Sm in Str-S wild-typestrains (str+), but the efficiency is decreased inStr-R mutants (104). Again, several differentStr-R mutations were observed showing differentdegrees of restriction. Similar observations weremade by Apirion and Schlessinger (8). (iii) Re-striction of Sm-induced misreading in vitro. Sm-induced misreading of synthetic messenger RNAin vitro is high when ribosomes from Str-S wild-type strains are used, but is very low when ribo-somes from Str-R mutants are used (60). (iv)Other properties. Couturier, Desmet, and Thomas(46) observed that many of the Str-R mutants(and some Str-D mutants) obtained from E. coliK-12 strain C600 showed a reduction in theirgrowth rate, inhibition of restriction of phagegrowth by prophage PI, and stimulation of lyso-genization of a clear-plaque mutant, c72. Theyobserved that each Str-R mutant expressed eitherall three of these characteristics or none. Althougha unified explanation for all three characteristics isnot readily apparent, the reduced growth ratesuggests that some Str-R mutations are delete-rious, probably because of a reduced efficiencyin protein synthesis. The pleiotropic effects ofStr-D mutations were observed by several workers(24, 101, 160, 266, 302, 303). In this case, a de-tailed explanation of the complex observedphenomena could not be made except that mostof the phenomena were interpreted on the basisof altered ribosome structure. Presumably, altera-tions in a single 30S protein, Plo, caused the va-riety of phenotypes and these phenotypes mustultimately be explained on this basis.

Gorini and his co-workers have studied therestriction of genetic as well as phenotypic sup-pression caused by Str-R mutations. They founda direct correlation between the degree of re-striction of Sm-induced suppression and the de-gree of restriction of genetic suppression bysuppressor tRNA (103). They also showed thatStr-R mutations restrict the genetic suppressionof missense mutations. Again, the degree of re-striction was correlated with that of the two typesof restriction described above.

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They also observed that the degree of restric-tion of suppression of a given amber codon by agiven str mutation differs for the three ambersuppressors tested (Sul, SuIl, and SuIllI), butthat there was no difference between allelic amberand ochre suppressors (SuII, and SuF, or SulIland SuG). Since the three amber suppressorstested (Sul, SuIT, and SuflI) involved mutatedtRNAs with different structures but carrying thesame anticodon, whereas the allelic amber andochre suppressor tRNAs differ only in their anti-codons, they could conclude that some aspect oftRNA structure not involving the anticodon de-termines the extent of restriction imposed by theStr-R mutation.From these observations, Gorini suggested that

the protein controlled by the str locus is not dir-rectly involved in the codon-anticodon interactionbut is involved in a process which facilitates thetRNA-ribosome binding process in a nonspecificway (103). Since the amount of suppressor tRNAin bacterial cells is very small, a mutational alter-ation affecting the general efficiency of tRNAbinding would cause a general reduction in theefficiency of genetic suppression. The fact thatsuppression by a ribosomal mutation at the strlocus in the absence of Sm has never been ob-served supports the conclusion that this locus isnot involved in the direct codon-anticodon inter-action (8).

Rosset and Gorini (267) looked for a ribosomalmutation which does cause suppression in theabsence of Sm. They started with a strain whichcarried both an amber mutation in the argF locusand a Str-R mutation restricting Sm-induced phe-notypic suppression. Fromthis strain, they selectedmutants which could overcome the restrictioneffect of the Str-R mutation and grow on a mini-mal medium containing Sm. One of these mutantscarried the desired mutation (ram, ribosomalambiguity), and when the Str-R mutation wasreplaced by its wild-type allele (str+) the mutantshowed suppression of all three types of nonsensecondons in the absence of Sm. Furthermore, theribosomes isolated from the ram mutant showedextensive misreading of synthetic mRNAs, andthis altered property was shown to reside in the30S subunit. The ram mutation was shown tomap very close to the spc (spectinomycin sensi-tivity) locus and was shown to be distinct fromthe str locus. Thus, a 30S ribosomal componentcontrolled by the ram gene must play an impor-tant role in maintaining translational fidelity.Rosset and Gorini (267) pointed out the similarityof the effects caused by the ram mutation to thosecaused by Sm. They also showed that the am-biguity caused by both the ram mutation and by

Sm is antagonized in vivo and in vitro by addi-tional mutations at the str locus. Thus, the re-lationship between the str locus and the ram locusclosely parallels the relationship between theproteins P1o and P7, as observed in the reconstitu-tion work described before. The protein alteredby the str locus is Plo. The protein altered by theram mutation has not yet been identified. How-ever, since P7 is the only protein whose omissioncaused a pronounced increase in misreading inthe reconstitution experiments, it is tempting topredict that it is protein P7 which is controlled bythe ram locus (234).From the work on Str-R mutations and the

ram mutation described above, Gorini (103) sug-gested that Sm acts on the ribosomal proteincontrolled by the ram gene, rather than on thestr protein, to cause misreading. He also sug-gested that the function of the str protein is tofacilitate tRNA binding in a nonspecific way withthe unavoidable consequence of increased transla-tional errors. Indeed, in vitro reconstitution workon the function of ribosomal proteins supportsthe conclusion that it is another protein (P7) andnot the str protein (Plo) which strengthens thespecific codon-anticodon interaction and thus isessential for maintaining translational fidelity.Also, a major consequence of the presence of thestr protein (Plo) in the 30S subunit is the increasederror frequency. However, in vitro reconstitutionexperiments did not reveal any significant de-crease in the synthetic mRNA-directed tRNAbinding ability of 30S particles when Plo wasdeleted. Rather, the reconstitution experimentsrevealed an important function of Plo in chaininitiation.

Thus, the function of Plo suggested by Gorini'sinterpretation of in vivo experiments has not beensupported by in vitro experiments. However, itshould be pointed out that the tRNA-bindingassay used in the reconstitution studies (234, 250)was carried out in the presence of high Mge andin the absence of T factors (see 164a, 173, 257).A tRNA-binding assay in the presence of T fac-tors would be better for testing the validity of theabove suggestion. It should also be pointed outthat, in the reconstitution experiments, the strprotein (Plo) was the only protein whose omissioncaused any significant decrease in Sm-inducedmisreading (234). Most of the other protein-deficient particles, except some showing verylittle synthetic activity, responded to Sm. Thisimplies that most of the other proteins, includingP7, are not required for the expression of theambiguity induced by Sm. Moreover, Plo is es-sential for the binding of radioactive dihydro-streptomycin, although binding to isolated P1o

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could not be demonstrated (250). Thus, contraryto the above suggestion made from in vivo experi-ments, the in vitro experiments indicate that thesite of action of Sm is P1o itself. It appears thatPlo is a part of the ribosomal structure whichinteracts with the initiation factors, that is, apart of the "initiation site." This structure mayalso correspond to the so-called A site in the 70Sparticle which is involved in the T factor-depend-ent binding of aminoacyl-tRNA. In vitro experi-ments done by Modolell and Davis (202, 203)also support the conclusion that Sm appears toact on the so-called A site involving Plo. However,more studies are needed to explain all the experi-mental observations made both in vivo and invitro.

Interaction among ribosome mutations. Twokinds of interactions among ribosome compo-nents can be considered. One is related to theassembly process and was clearly demonstratedin the in vitro assembly of 30S subunits. Forexample, it was found that protein P13 cannotbind to 16S RNA directly, but can bind to it inthe presence of P5 (see Fig. 3 and Assembly Proc-ess). A mutational alteration in P13 may decreaseor abolish the binding of P13, leading to a partialor complete defect in the assembly reaction. Asubsequent mutational alteration in P5 couldenhance or cancel the assembly defect due to theoriginal mutational alteration in P13. The secondkind of interaction is an interaction amongproteins that are not obviously interdependentin the assembly process. Such interaction was alsoindicated in reconstitution studies, although lessclearly. The presence of several proteins whichare not required for ribosomal assembly and areonly stimulatory in functional assays could sug-gest a cooperative functional interaction amongribosomal proteins (see Reconstitution of Ribo-somes). For example, P7 and P10 were both shownto have functions related to translational fidelity,but acting in an opposing manner. Thus, inter-action among certain ribosomal mutations af-fecting these proteins can easily be conceived.There are many published experiments sug-

gesting interactions among ribosome mutations(see reference 281). Here we shall mention a fewwell-studied cases. The first is the interactionbetween Str-R mutations and the ram mutationwhich we have already discussed above. Thesecond is related to the suppression of Str-Dmutations. Hashimoto (117) showed that most ofthe mutations from streptomycin dependence toindependence are not true reversions but are dueto a second mutation very closely linked to theoriginal mutation. Brownstein and Lewandowski(32) showed that ribosomes from such a "rever-

tant" (suppressed Sm dependence, or Str-SD) aresensitive to Sm in an in vitro polypeptide syn-thesizing system, in copntrast to the ribosomesfrom the parental Str-D strain which are resistantto Sm in vitro. In similar experiments with otherstrains, it was shown that 30S ribosomal subunitsfrom Str-SD strains are sensitive to Sm in vitro,in contrast to 30S subunits from the parentalStr-D strains (Birge and Kurland, personal com-munication; Guthrie, Nashimoto, and Nomura,unpublished data). Thus, the second mutationalalteration suppressing the original Str-D muta-tion is also in the 30S ribosomal particle. Re-cently, with another Str-SD strain, Apirion andhis co-workers observed an alteration in the gelelectrophoretic pattern of 30S ribosomal proteins(9a). Their results suggest that the componentaltered by the second mutation is not Plo, whichis the component altered by the original Str-Dmutation. Precise identification of the secondaltered protein and the study of the interactionbetween this protein and the protein (Plo) from aStr-D strain by means of in vitro reconstitutionsystems should be interesting.The third case of an interaction between muta-

tions is that between Str-R and Spc-R mutations.Spectinomycin is a strong inhibitor of proteinsynthesis in E. coli both in vivo and in vitro. Itwas shown that sensitivity and resistance to thisdrug are properties of 30S ribosomal subunits(4). The genetic locus (spc) conferring resistancemaps close to the str locus (3, 4, 79, 348). Thecomponent altered by the mutation to spectino-mycin resistance (Spc-R) was recently identifiedas P4, using the reconstitution technique (22).Thus the genes str and spc control two differ-

ent 30S ribosomal proteins, Plo and P4, respec-tively. Kuwano, Endo, and Ohnishi (158) ob-served that Spc-R mutations restored the sup-pressor activity of the Sull amber suppressor toStr-R strains in which the suppressor activity hadbeen restricted. Spc-R mutations by themselvesdo not have any effect on suppression. Also,spectinomycin does not cause phenotypic sup-pression or misreading as does Sm (4). Thus, itappears that the spc protein (P4) interacts func-tionally with the str protein (Plo) in the ribosome.No experiment designed specifically to test thisin vitro has been performed.

There also seems to be an interaction betweenSpc-R mutations and mutations to neomycinresistance (Nek-R). Apirion and Schlessinger (9)introduced a Nek-R mutation into cells carrying aSpc-R mutation and observed that some of thedoubly mutant strains were phenotypically sensi-tive to spectinomycin. Protein synthesis in cell-free extracts from such double-mutant strains

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was shown to be sensitive to spectinomycin. Thus,the alteration in a ribosomal component causedby the Nek-R mutation appears to cancel theeffect of the alteration in the P4 protein caused bythe Spc-R mutation. However, the componentaltered by the Nek-R mutation has not beenidentified.

Clustering of ribosomal mutations. In additionto the str and spc genes, the K locus is known tocontrol a 305 ribosomal protein. Leboy, Cox, andFlaks (162) found that a 30S ribosomal protein(P5) from E. coli strain K-12 is different from thecorresponding protein from other E. coil strainsin its electrophoretic mobility on polyacrylamidegels. The difference between these two proteinswas confirmed by chemical analysis (18, 317).This character (K character) can be transferredfrom K-12 strains into B strains by PI transduc-tion by using streptomycin as a selective marker;hence, the genetic determinant, the K locus, isvery closely linked to the str locus (162, 188).Thus, genetic determinants of three 30S proteinsappear to be linked together very closely. Em-ploying a similar approach, that is, using electro-phoretic mobility differences between some of theE. coil 30S proteins and the corresponding pro-teins of Salmonella typhosa as genetic markers,Sypherd and his co-workers (241, 319) showedthat the genetic determinants for at least two moreE. coli 30S proteins also map in the region nearthe str locus. Several other mutations which weresuggested to affect 30S proteins also map nearthe str locus, but these mutations have not beendefinitely characterized. It is still premature toconclude that all the 30S ribosomal proteins areclustered in one region of the E. coil chromosome.In fact, it has recently been found that althoughkasugamycin resistance in E. coli appears toaffect a 30S ribosomal component, the resistance(ksg) maps at a distance from the str region,namely, near leu (291). Nevertheless, an extensionof mapping studies to include all the ribosomalproteins should be informative in relation to thegenetic control of the biosynthesis of ribosomecomponents and ribosome assembly.

Mutations affecting 505 subunits are less wellstudied, and hence the question of clustering ofgenes for 50S ribosomal proteins will not be dis-cussed here. [Refer to the review by Schlessingerand Apirion (281) for available information.]

Similarly, the location of the genetic determi-nants for ribosomal RNA is not well establishedin E. colf. Some studies suggest that the ribosomalRNA genes in E. coli are not located in the strregion (79, 319), whereas other studies suggestthe contrary, namely, that the rRNA genes arelocated in the str region (55). In B. subtilis, the

ribosomal RNA genes are located in two chro-mosomal regions, the major and minor regions.The major region is closely linked to a strrgene which codes for a 305 ribosomal protein(66, 99, 240, 287, 288).

MECHANISM OF ASSEMBLY OF 30SRIBOSOMAL PARTICLES IN VITRO

The complete reconstitution of 305 ribosomalsubunits has allowed the study of the mechanismof assembly of ribosomal particles in vitro. Thefirst important observation made with thissystem was that the reconstitution is independentof the presence of additional macromolecularstructures, such as enzyme, membranes, pre-existing ribosomes, etc., or of an added energysource. This observation led to the conclusionthat the in vitro assembly of active 30S subunitsis a spontaneous process and that the informa-tion for correct assembly is completely containedin the structure of the ribosomal components.However, this does not mean that the assemblyprocess in this system is exactly the same as thatin vivo. For example, as will be described in thenext section, addition of at least some of theproteins to RNA in vivo appears to take placewith submethylated immature "16S" RNArather than the fully methylated mature 165RNA that is used in the in vitro reconstitution.Nevertheless, studies on the mechanism ofribosome assembly in vitro have revealed severalimportant features of the construction of acomplex cellular organelle and have suggestedpossible mechanisms for the biosynthesis of 30Sribosomal particles in vivo.

Conditions Necessary for Total ReconstitutionSeveral factors influencing the efficiency of

reconstitution were examined by Traub andNomura (332). Use of rather high ionic strengthwas found to be important. The optimal ionicstrength was found to be 0.37. At a low ionicstrength no measurable reconstitution tookplace. The high ionic strength may be necessaryto prevent the electrostatic attraction betweenpositively charged ribosomal proteins and nega-tively charged ribosomal RNA that would leadto nonspecific RNA-protein aggregation. Be-yond the optimal ionic strength of 0.37, the yieldof functionally active material decreased withincreasing ionic strength. This inhibition is pre-sumably due either to a weakening of specificinteractions by a high salt concentration or tothe formation of an inactive, highly compactribosomal RNA or intermediate ribonucleo-protein structure.Another important factor was the tempera-

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ture of the incubation mixture. At 10 C or below,the reconstitution did not proceed at measurablerate. There was a sharp rise in the extent of thereaction between 20 and 40 C; the maximal ratewas obtained at 40 C.

Kinetics of the Assembly and the Presence ofIntermediates

The kinetics of the reconstitution process wasexamined at various temperatures. It was foundthat the formation of active 30S ribosomalparticles follows first-order kinetics. Since allreactants are present in the reconstitution mix-ture in approximately equivalent amounts, itwas concluded that there are several steps in theassembly reaction and that the rate-limiting stepis unimolecular. The activation energy for thisrate-limiting step was calculated from the tem-perature dependence of the rate of reconstitu-tion. It was calculated that the reaction has ahigh enthalpy of activation (37 kcal/mole) anda positive entropy of activation [50 cal permole per degree (C) of temperature].

Several lines of experimental evidence showedthat this rate-limiting reaction does not representa structural change of free 16S RNA, but rathera structural rearrangement of an intermediateparticle with some but not all of the ribosomalproteins bound to the 16S RNA. On incubationof the reconstitution mixture at 0 C, no active30S particles were produced. Examination ofsuch reaction mixtures showed that neither free16S ribosomal RNA nor inactive 30S particleswere present; instead, new particles (21S "RIparticles," for reconstitution intermediate) ac-cumulated. These particles were isolated bycentrifugation and shown to be deficient inseveral protein components ("S proteins")which are recovered from the supernatantfraction. The proteins contained in the RIparticles were called RI proteins. RI particleswere functionally inactive. Neither restorationof their functional activity by S proteins norefficient binding of S proteins took place at 0 C;incubation at a higher temperature was required.However, heating RI particles alone at 40 Cfor 20 min produced particles (called "RI*particles" for activated reconstitution inter-mediates) which were now capable of binding Sproteins at 0 C to become functional 30S par-ticles. From these results, it was proposed thatthe reconstitution takes place in a stepwisefashion:

16S RNA +RIproteinsI RI particles

RI* partice +s proteins 305 ti

The reaction RI particles RI* particles is therate-limiting unimolecular reaction and repre-sents a structural rearrangement with a highactivation energy. Although the protein distribu-tion in the S and RI fractions was not studied inany detail, gel electrophoretic analyses indicatedthat most of the proteins which can be removedfrom 30S subunits by treatment with 5 M CsCl("split proteins") were contained in the S frac-tion. However, some split proteins were found inthe RI fractions. The proportion of RI* particleswhich could be converted to functional 30Sparticles ranged only from 20 to 40%. It ispossible that the isolated RI particle preparationwas not homogeneous but contained severaldifferent molecular species, as suggested bypolyacrylamide gel analyses. This would meanthat only a small fraction of the particles in theRI preparation were real functional intermedi-ates. It is also possible that the actual inter-mediate is produced only by dissociation of someproteins from the RI particles during the heatingstep. Thus, the rigorous identification of theintermediate and the rate-limiting rearrangementreaction must await further studies. Never-theless, the proposed scheme must be basicallycorrect, and reconstitution studies using theseparated protein components of ribosomesshould soon allow an unequivocal identificationof the intermediate in the proposed scheme.

Sequential and Cooperative Nature of the Assembly

Preliminary experiments were performed tofind the number of sites or groups of sites perRNA chain which can bind ribosomal proteinsindependently of each other (i.e., without co-operativity) under in vitro conditions (237).Reconstitutions were performed under thestandard conditions but with various amountsof the protein mixture and a constant amountof RNA. If there are many independent protein-binding sites on the RNA chain, the fraction ofRNA with a complete set of proteins would bevery small in the presence of excess amounts ofRNA. That is, excess RNA would inhibit theformation of 30S particles containing a completeset of proteins by forming various kinds of in-complete RNA-protein complexes. On theother hand, one could imagine a situation inwhich all the protein components interact withRNA as one group, forming some active ribo-somes and leaving excess RNA as free RNA.The experiments showed that the assembly re-action is highly cooperative and that the forma-tion of active 30S particles with a complete set of"essential" ribosomal proteins is the dominantreaction under the standard reconstitution con-

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ditions. The stable accumulation of manypossible inactive particles which are deficient insome essential ribosomal proteins did not occur(237).To explain the cooperative nature of the as-

sembly reaction and to determine the sequenceof events during reconstitution, more systematicstudies have been initiated in our laboratory(201). We first asked which of the 21 isolatedribosomal proteins interacts directly with 16Sribosomal RNA under reconstitution conditions(ionic strength, 0.37; Mg+2 concentration, 0.02 M;and temperature, 40 C). Each of the purifiedribosomal proteins was mixed separately with16S RNA and was incubated. The reactionmixture was then analyzed by sucrose gradientcentrifugation. It was found that three proteins(P4a, P4b, and P14) could combine with 16SRNA in a roughly stoichiometric way. Threeother proteins (P5, Pg, and P10.) showed weakbinding, whereas none of the other 15 proteinsshowed any significant binding. Next, each ofthe 18 proteins which showed either weak bind-ing (P5, P9, and Ploa) or no binding to the 16SRNA was examined for its ability to bind tothe complex consisting of 16S RNA, P4a; P4b,and P14. Three proteins, P5, P9, and Pioag nowshowed an almost quantitative binding, but noneof the other 15 proteins was found to bind. Thebinding of P9 to the complex was dependent onthe presence of either P48 or P14, but not on thepresence of P4b. In the presence of both P48and P14, the maximal binding of P, to the com-plex was observed (Fig. 3). Next, each of theremaining 15 proteins was examined for itsability to bind the complex consisting of 16SRNA, P4a, P4b, P14, P9, and Ploa. Only P4 showedgood binding. The binding of P4 did not dependon the presence of P1o0, but depended on thepresence of the other four proteins in addition to16S RNA. Therefore, it was concluded that P4can join the complex with the aid of both P4b andP9, the latter (P,) being dependent on P48 andP14 (Fig. 3). In this way, the order of addition ofall the 30S ribosomal proteins to the 16S RNAwas examined. Although somewhat ambiguousresults were obtained for the later part of the"sequence," the initial sequence was clearlydetermined (see Fig. 3). Sometimes, significantjoining of two proteins to a ribosomal inter-mediate can take place only when both proteinsare present at the same time. (For example, Pi1and P6 can bind efficiently to the complex con-taining 16S RNA, P5, and P13 only when bothare present simultaneously.) Although it isdifficult to examine interdependence amongprotein components by making all the possible

P2Pl5

r-I

P3a

FIG. 3. Assembly map of 30S ribosomal proteins.Arrows between proteins indicate the effect of a pro-tein on another protein whose binding it helps. A thickarrow indicates a major contribution. The following areexamples of use. Thick arrow from 16S RNA to P4a:P48 binds directly to 16S RNA in the absence of otherproteins. Thin arrow from 16S RNA to PF: P5 bindsweakly to 16S RNA in the absence of other ribosomalproteins. Thin arrows pointing towards P, from P4w,Pab, P14, P8 and Pi: the latter proteins all help thebinding of P, to RNA. Thick arrow from P, to Pis:in the absence of P5, Pis cannot be bound to complexescontaining 16S rRNA even in the presence of all otherproteins. Arrow to P7 from large box (outlined withdashes): P7 binding depends on some of the proteinsenclosed in the box; it is not exactly known whichones. P2 binding: to avoid complication, we have notincluded location of P2 in the map. Plo binding require-ments are not known. P1 and Pa. do not bind under theconditions used, and P1 -is probably a nonribosomalprotein. This map is from the work of Mizushima andNomura (201).

combinations of proteins in this way, our bestapproximation of the general pattern of inter-dependence is represented by the scheme shownin Fig. 3. Only a few proteins can bind to asignificant extent to 16S RNA in the absence ofany other proteins. The remaining proteins can-not interact with RNA without the simul-taneous presence of some other proteins.There are several comments to be made in re-

lation to the above results. (i) Proteins whoseomission gave a drastic effect on the sedimenta-

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tion pattern in reconstitution experiments (seeReconstitution of Ribosomes and Table 2), namelyP4ar P4b, P5, P9, and P8, were found in the earlypart of the reconstitution sequence. All theseproteins were shown to be essential for thebinding of several other proteins. Although P14interacts with RNA directly, its omission didnot show a drastic effect on the sedimentationpattern of reconstituted particles. (ii) The in vivosignificance of the sequence, or interdependence,of binding of proteins elucidated in vitro wasexamined. As will be described in a later section,there is a cold-sensitive mutant of E. coli whichaccumulates a particle with a sedimentationcoefficient of 21S, which is probably a pre-cursor of 30S subunits or related to them. Analysisof protein components of the 21S particles showedthat nine proteins found in these particles areall 30S ribosomal proteins, and that all but one(P3a or P3b or P3, not definitely identified) areproteins involved in the early part of the re-constitution sequence (see Fig. 3). No incon-sistency with the in vitro sequences was observed(Nashimoto and Nomura, unpublished data).Such strong correlation between in vivo andin vitro results strengthens the significance ofthe assembly mechanism elucidated in vitro.(iii) As described above, binding of many of theproteins is dependent on the presence of someother proteins. For example, the binding of P4is dependent on the presence of both P4b andP9, and the binding of P9 is in turn dependenton P4a or P14, or both. However, this does notnecessarily mean that P4 has no direct interactionwith 16S RNA. The RNA may still have aspecific site for P4, but the binding of P4 to thissite may be very weak and may require the inter-action of P4 with other proteins to stabilize thebinding. Alternatively, a specific site for P4on the RNA may be created by some confor-mational alteration of the RNA induced by thebinding of other needed proteins to the RNA.Of course, it is also possible that P4 has in fact nodirect physical interaction with the RNA. Onedirect approach to solving this problem wouldbe to isolate various specific RNA-proteincomplexes in the above experiment, to removethe free uncomplexed part of the RNA chain byribonuclease digestion, and then to examinewhich protein has protected which part of theRNA. It should also be pointed out that thespecific interdependence among proteins revealedin the experiments described above suggests ahighly specific topological relationship amongvarious ribosomal components in the three-dimensional ribosome structure.

BIOSYNTHESIS OF RIBOSOMES

Biosynthesis of rRNATemplate for rRNA synthesis. Although some

sort of "self replication" of rRNA was con-sidered in the past, it is now definitely establishedthat rRNA is first synthesized on the DNAtemplate. It then undergoes some post-tran-scriptional modification to become the "ma-ture" 16S or 23S rRNA that is found in theribosome. The evidence for DNA as the directtemplate for rRNA synthesis is as follows.(i) Yankofsky and Spiegelman (355-357), andsubsequently many other workers, showed thatrRNA can form specific RNA-DNA hybridswith denatured homologous DNA. This showsthat the ultimate template for rRNA synthesisis DNA, but does not prove that it is the immedi-ate template. (ii) Actinomycin D and rifampin,which are specific inhibitors of DNA-dependentRNA synthesis, inhibit the rRNA synthesis inbacterial cells (167, 328). (iii) Mutants of E.coli which are resistant to rifampin or relatedantibiotics have normal rRNA synthesis in thepresence of the antibiotics. It was demonstratedthat the DNA-dependent RNA polymerase iso-lated from these mutants was altered and wasresistant to the antibiotics in an in vitro ac-tivity test (328, 360). Thus DNA-dependentRNA polymerase must be the major enzymeinvolved in the synthesis of rRNA as well as ofother RNAs in vivo.The synthesis of rRNA or fragments of rRNA

in vitro was demonstrated recently by Pettijohn(253). He isolated a crude preparation containingDNA and RNA polymerase, and showed thatthe RNA synthesized includes rRNA or frag-ments of rRNA as judged by DNA-RNA hy-bridization. Since DNA fragments consisting ofmostly rRNA genes have now been isolated(239), it should be possible to study rRNA syn-thesis in a better-defined system. Such an in vitrosystem should be useful not only for confirmingDNA as the template for rRNA, but also forfinding out possible factors involved in the regula-tion of rRNA synthesis in vivo.

Maturation of rRNA. It is known that ineukaryotic systems there exist large precursorRNA molecules, 45S RNA, which undergoseveral cleavage processes and eventually produceboth 18S and 28S rRNA (see 58). In bacteria,by contrast, there do not appear to be any suchlarge precursor rRNAs which produce both 16Sand 23S rRNA. However, two kinds of experi-ments showed that both the 16S and 23S rRNApresent in ribosomes are not primary transcriptionproducts, but are derived from some sort ofprecursor rRNAs. First, rRNAs synthesized in

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the presence of chloramphenicol (CM) or duringamino acid starvation in relaxed bacterial strainswere examined. These rRNAs synthesized in theabsence of protein synthesis exist as ribonucleo-protein particles (CM particles or relaxed par-ticles) in cell extracts. It was found that therRNAs extracted from these particles are de-ficient in methylated nucleotides, and that thesmaller rRNA found ("17S" RNA) sedimentsslightly faster than mature 16S rRNA (65, 102,150, 318). Second, the rRNAs in ribosome pre-cursor particles identified by a pulse-labelingtechnique were shown to be similar to the rRNAsin CM particles (245). Recently, more refinedRNA separation techniques such as polyacryl-amide gel electrophoresis were used to examinethese precursor forms of 16S and 23S rRNA(1, 57, 119). Both precursors have electrophoreticmobilities slower than the mobilities of theirmature forms; the precursor to 16S rRNA(precursor 16S rRNA or "17S" rRNA) can beresolved from its mature counterpart, whereasthe two 23S forms are less well separated. Dueto the difference in electrophoretic mobilitybetween precursor 16S rRNA and mature 16SrRNA, their different sedimentation behaviorcannot be explained by a conformation differencealone and must be due also to a molecularweight difference. The molecular weight of pre-cursor 16S rRNA was estimated to be greaterthan that of mature 16S rRNA by 50,000 daltonsin E. coli (1) and by 130,000 daltons in B. sub-tilis (119), whereas the molecular weight ofprecursor 23S rRNA was estimated to be greaterthan that of mature 23S rRNA by 50,000 to100,000 daltons (119). A kinetic analysis of thepulse-labeling data suggests that both the identi-fied 16S precursor and the 23S precursor areprimary transcription products, whereas mature16S rRNA and mature 23S rRNA are not (1,119). Similarly, the 5S RNA present in 50Sribosomal subunits does not appear to be aprimary transcription product (118). As in theformation of mature 16S and 23S rRNAs, theformation of "mature" 5S RNA is inhibited byCM in B. subtilis as judged by the technique ofgel electrophoresis. Recently, "CM-5S RNAs"synthesized in E. coli in the presence ofCM wereisolated and a difference in terminal sequencefrom that of normal 5S RNA was demonstratedby Forget and Jordan (82, 209). Whereas thenucleotide sequence of the 5'-terminus of normal5S RNA is UG-, there were three alternatesequences for the 5'-terminus of CM-5S RNA:UUG-, UUUG-, or AUUUG-.

Another question related to rRNA maturationis whether the precursor 23S rRNA is formedby dimerization of smaller "165 rRNA." Such

a hypothesis has been advocated especially byMangiarotti et al. (180). They analyzed thekinetics of rRNA synthesis in slowly growing(doubling time, 120 min) fragile E. coli cellsand concluded that the time required to completethe 16S precursor rRNA chain is the same asthat required for the completion of the 23Sprecursor rRNA chain. A similar observationwas made by Adesnik and Levinthal (1). Man-giarotti et al. (180) also found that for shortpulses in pulse-labeling experiments, a consider-able portion of newly synthesized rRNA waspresent as "precursor ribonucleoprotein par-ticles" sedimenting more slowly than 26S. Thenewly synthesized rRNAs, upon deproteiniza-tion, sedimented more slowly than 16S, andnever between 16S and 23S. Since these rRNAscompete with purified mature 16S and 23S rRNAin DNA-RNA hybridization, it was concludedthat they are 16S and 23S rRNA precursors.From these observations, it was suggested thatcomplete "23S" rRNA molecules are formedby the joining of two molecules of "16S" RNA.Such a hypothesis is also consistent with thedata obtained by Fellner and Sanger (76) thatall the methylated oligonucleotides obtainedafter Ti ribonuclease digestion of mature 23SrRNA are contained in an amount correspondingto 2 moles per mole of 235 rRNA. However, thehypothesis needs more rigorous experimentalproof before it can be accepted as fact.There are two features evident in the matura-

tion process. One is methylation and the otheris cleavage of some portions of RNA chains.The sequence of events is unclear. Some avail-able data weakly suggest that the size of rRNAbecomes normal prior to methylation (40)and, conversely, that methylated rRNA whichstill has the size of precursor rRNA can exist(316). It is necessary to ask what sort of mecha-nism ensures the specific cleavage of precursorrRNA to produce homogeneous mature rRNAand how methylating enzymes work to producethe specific methylation. Any explanation musttake into account the fact that methylation andcleavage both are inhibited by CM. Two ex-planations are possible. (i) It is the binding ofsome ribosomal proteins that creates the specificstructures which act as substrates for methylatingenzymes or hypothetical specific nucleases.Thus, inhibition of the synthesis of ribosomalproteins by CM would lead to inhibition ofboth cleavage and methylation. (ii) Both cleavageand methylation are catalyzed by the ribosomalproteins themselves, and after completion of thereactions, the proteins are incorporated into theribosomal structure. However, there is no ex-

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perimental evidence to support either of theseexplanations.

Several possibilities can be considered for thephysiological role of precursor rRNAs. Onepossibility is that the interaction of ribosomalproteins with rRNA in the assembly processcan take place only on the precursor rRNAand not on the mature rRNA. In vitro reconsti-tution of 30S ribosomal subunits from proteinsand mature 16S rRNA (331) makes this possi-bility unlikely. However, it is still conceivablethat the in vivo rate of ribosome assembly mightbe faster with precursor rRNAs. In vitro re-constitution experiments using the precursor16S rRNA (obtained from "relaxed particles")have not indicated such a rate difference so far(Nepokroeff and Nomura, unpublished data).Another possibility is that the precursor rRNAserves as a template for the synthesis of ribosomalproteins, whereas the mature rRNA does not.A third possibility is that the extra segments ofRNA present in precursor rRNA have someunknown regulatory function related to thetranscription of rRNA or the assembly of ribo-somes. The presence of extra RNA segments inprecursor rRNAs and their specific excision is afeature common to both eukaryotic and pro-karyotic cells. Perhaps the extra RNA segmentshave an important role which is the same ineukaryotic and prokaryotic systems.

Regulation of rRNA synthesis. The rate ofbacterial growth and hence the rate of proteinsynthesis are proportional to the number ofribosomes per cell (142, 143, 177, 226, 276).The amount of rRNA and hence the number ofribosomes per cell depend on the type of growthmedium. When a culture of bacteria is shiftedfrom a minimal medium to a rich medium, therate of rRNA synthesis is accelerated almostimmediately, whereas the overall rate of synthesisof proteins and DNA is accelerated only after aconsiderable time lag. Conversely, when a cul-ture is shifted from a rich medium (e.g., a glucosemedium) to a poor medium (e.g., a succinatemedium) synthesis of ribosomal RNA is tem-porarily inhibited and then is resumed at therate characteristic for the poor medium (143,144, 226, 227).The rate of synthesis of ribosomal proteins

also changes in response to the type of growthmedium and parallels the change in the rate ofrRNA synthesis (278). Thus, there is a regulationof the rate of synthesis of both rRNA and ribo-somal proteins in response to environmentalconditions. The mechanism of this regulation isunknown. It does not appear to be the same asthat involved in the regulation of RNA synthesisby amino acids, since relaxed strains respond to

changes of media in the same way as stringentstrains (225).

In most bacterial strains, the synthesis ofrRNA requires the presence of amino acids.The exact mechanism of this regulation by aminoacids is still unknown. Because an extensivereview on this subject appeared recently (67),we shall limit ourselves to mentioning some ofthe possible mechanisms relevant to the generalregulation of rRNA synthesis.The hypothesis that uncharged tRNA is a

repressor of rRNA synthesis (155, 311) is nowconsidered unlikely for several reasons (67).For example, no good correlation was foundbetween the amount of uncharged tRNA meas-ured inside cells and the rate of rRNA synthesis(73).

Since there is a good correlation between thenumber of ribosomes or amount of rRNA presentand the rate of protein synthesis, it could besupposed that the rate of rRNA synthesis isregulated by some mechanism sensitive tochanges in the state of the protein synthesizingapparatus. It was suggested that free ribosomesor ribosomal subunits themselves are the re-pressors of rRNA synthesis (215, 264). The initialobservations leading to this suggestion werethat polysomes break down upon amino acidstarvation in stringent strains, but not in relaxedstrains. Although these observations were notconfirmed by more extensive experiments (84),the hypothesis cannot be excluded. Shih andhis co-workers (286) observed that inhibition ofpolypeptide chain initiation by trimethoprimresults in the cessation of rRNA synthesis instringent strains but not in relaxed strains. Aconsequence of the inhibition of polypeptide-chain initiation is a change in the state of ribo-somes and polysomes, and therefore it is con-ceivable that some state of free ribosomes isconnected with the regulation of rRNA synthesis.Further development of cell-free systems whichsynthesize rRNA (253) may be useful for examin-ing such a hypothesis.Another hypothesis proposed to explain

regulation of RNA synthesis in general is theobligatory coupling between protein synthesisand RNA synthesis proposed by Stent (309,310). This hypothesis states that the attachmentof ribosomes to nascent mRNA being synthe-sized on the DNA template stimulates thefurther synthesis of mRNA. In the case of regu-lation of rRNA synthesis, this hypothesis mustassume that nascent precursor rRNA acts as atemplate for some of the ribosomal proteins.This assumption will be considered in the nextsection.One could also propose that the regulation of

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rRNA synthesis is mediated by some proteins,possibly ribosomal proteins, which bind directlyto nascent ribosomal RNA and somehow pro-mote its completion. In this connection, it isinteresting to note the recent experiments byGoodman, Manor, and Rombauts (100). Usingamino acid-requiring stringent and relaxedstrains, they found that preferential synthesisof a few (at least three) ribosomal protein speciestakes place in the relaxed strain during aminoacid starvation, but not in the stringent strain.Even though total protein synthesis was reducedto 3% of the normal level by the amino acidstarvation, and the synthesis of most of the ribo-somal proteins was also reduced drastically, thesynthesis of these special ribosomal proteinscontinued at about the normal level. It is clearlyimportant to identify these proteins and to studytheir function both in vivo and in vitro.

In eukaryotic systems, it is known that selec-tive replication of rRNA genes takes place undercertain conditions. In developing oocytes of frogs,salamanders, and other animals, a huge increasein the number of rRNA genes relative to that inother tissue cells was demonstrated by RNA-DNA hybridization methods (29, 89). The rRNAgene copies accumulate in the many nucleolifound in oocytes. This selective amplification ofrRNA genes is accompanied by a high rate ofrRNA synthesis during oogenesis. Thus, theamplification appears to be a special mechanismto increase the rate of rRNA synthesis. Inbacteria, it is not known whether such a mecha-nism exists. However, several copies of the rRNAgenes occur within the bacterial genome. Pos-sibly this increases the probability of the loopingout of a section of rRNA genes to form anepisome. Then an amplification of rRNA wouldresult from episome replication or enhancementof transcription in the episomal state, or fromboth. So far, no experiments have been conductedto test this possibility.The coordination of the synthesis of all the

ribosomal RNAs, 23S, 16S, and 5S RNA, isan important feature of the regulation of rRNAsynthesis. In eukaryotic systems, the presenceof a common 45S precursor RNA may insurethe coordinated synthesis of two of the rRNAs,18S and 28S RNA. However, even here the 5SRNA is synthesized on a section of the DNAtemplate which is not linked to the DNA con-taining the 18S and 28S cistrons; yet somemechanism insures the coordinated synthesisof the 5S and the other two rRNAs (28). Forexample, an anucleolate mutant of Xenopuslaevis is known to lack rRNA cistrons but tocontain normal amounts of 5S RNA genes, andyet the mutant fails to produce 5S RNA even

though it is capable of forming other mRNAs.In bacteria, there is no large precursor RNAcorresponding to 45S RNA, although cistronsfor 5S, 16S, and 23S RNA were shown to beclustered in B. subtilis (66, 240, 287). It is possiblethat these genes, although separate, may haveidentical promotor regions which utilize aa-like factor of RNA polymerase specific onlyfor these genes. This would lead to coordinatesynthesis of the ribosomal RNAs. However,some other mechanism, for example one in-volving the same repressor and operator for thevarious rRNA genes, is equally conceivable.

Perhaps studies of the extra RNA segmentspresent in rRNA precursors will give some clueas to the presence of regulatory mechanismsinsuring coordinated synthesis of all the rRNA.

Biosynthesis of Ribosomal ProteinsTemplate for ribosomal protein synthesis. The

possibility that rRNA codes for ribosomalproteins was originally supported by the experi-ments of Otaka and her co-workers (248). Al-though mature rRNA does not have any tem-plate activity in a cell-free protein-synthesizingsystem (230), Otaka et al. showed that a rRNApreparation isolated from cells exposed to CMcan stimulate amino acid incorporation. Similarexperiments were done by other workers (217,219). Furthermore, Holland et al. (127) demon-strated that even mature rRNA exhibits signifi-cant template activity when heated, especially inthe presence of neomycin. The structural restric-tions imposed by the maturation process mightthus act as a barrier to translation which can beremoved by treatments leading to a deformationof secondary structure, such as heating or interac-tion with aminoglycoside antibiotics.The hypothesis that nascent precursor rRNA

acts as a template for ribosomal proteins wasfurther elaborated by Nakada (219). He pro-posed that relaxed particles, which are incompleteribosomal particles synthesized in relaxed strainsduring amino acid starvation, are by themselvesable to synthesize the ribosomal proteins thatthey lack without the participation of completemature ribosomes. Although he showed aminoacid incorporation by relaxed particles, as wellas the conversion of relaxed particles to completeribosomes in the absence of ribosomes andmRNA, his experiments did not rigorouslyexclude the possibility of contamination of hisrelaxed particle preparation by ribosomes andmRNA. Subsequent experiments bore out thisobjection. Thus, Sypherd (315) and Manor andHaselkorn (183, 184) demonstrated that thebulk of messenger activity in their relaxed particle

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preparations was associated with a 10S RNAfraction. Muto, Otaka, and Osawa (218) ob-tained experimental results which indicated thatribosomes and polysomes, rather than in-complete relaxed particles, were the sites ofsynthesis of ribosomal proteins. More recently,Flessel showed that a polyribosome preparationfrom E. coli cells directed the synthesis of atleast 15 ribosomal proteins (C. P. Flessel, Ph.D.Thesis, Massachusetts Institute of Technology,1969; and manuscripts in preparation). Thusit seems that the mechanism of synthesis ofribosomal proteins is essentially the same asthat of all other proteins and takes place onmature ribosomes.However, whether the template for ribosomal

protein synthesis is precursor rRNA has stillnot been settled. Recent experiments by Muto(217) favor the hypothesis that 'the template isnascent precursor rRNA. Muto isolated andpurified rRNA from both CM particles and re-laxed particles. In a cell-free amino acid incor-poration system, these nascent rRNA prepara-tions were about one-third as active as f2 RNAin stimulating polypeptide synthesis. The possi-bility that this stimulatory activity was due tocontamination by mRNA could be excluded.Preliminary experiments showed that the 14C-labeled peptides made in vitro under the directionof nascent rRNA were similar to ribosomal pro-teins in their electrophoretic mobility on poly-acrylamide gel electrophoresis.The hypothesis that nascent rRNA codes for

ribosomal proteins has several attractive features.It provides a mechanism for the coordinatedsynthesis of rRNA and ribosomal proteins.Also, the assembly of ribosomal particles wouldbe facilitated by the physical proximity of theprotein products to rRNA. However, the hy-pothesis also presents some difficulties. Theprecursor 16S rRNA with a molecular weightof about 6 X 105 could code for only three tofive of the 30S ribosomal proteins. Similarly, theprecursor 23S rRNA could code for only 6 to10 of the 50S ribosomal proteins. Since 16SrRNA and probably 23S rRNA appear to belargely chemically homogeneous (74-76), rRNAcould not possibly code for all the ribosomalproteins. One could then postulate, however,that only a certain class of ribosomal proteinsis specified by the rRNA-perhaps the "as-sembly" proteins directly interacting with rRNA(e.g., P4a, P4b, P14, and possibly P5, P9, and P10,oin the case of 30S proteins). Such a mechanismmight serve to protect rRNA from degradationor to insure the proper assembly of the ribosome.Another problem with the hypothesis is that

it requires that the same nucleotide sequencewhich codes for a ribosomal protein also be ableto take on a suitable configuration for carryingout the structural and functional role of rRNAin the completed ribosome. However, due todegeneracy in the genetic code, several nucleo-tide sequences, all giving slightly different con-formations, could code for the same ribosomalprotein. It is conceivable that evolution couldhave selected for the most favorable sequence.Thus, a bifunctional rRNA is not impossible onthis basis.

Finally, according to Mangiarotti et al. (180)the nascent rRNA is never associated with poly-somes; yet ribosomal proteins are synthesizedon polysomes as described above. Recently,however, Fry and Artman (85) as well as Dahl-berg and Peacock (personal communication)presented experimental results indicating thepresence of significant amounts of nascent rRNAassociated with polysomes. Thus, the questionis not settled and further experiments are neces-sary. One approach would be to repeat Muto'sexperiments and characterize the protein productsmore rigorously and to identify the kinds of 305ribosomal proteins synthesized. Coupling tran-scription and translation steps in vitro, one couldalso perform similar experiments using as atemplate DNA which contains mostly rRNAgenes (239). Another approach would be thegenetic one. By mapping all the genes for 30Sribosomal proteins and comparing their mappositions with those of the 16S rRNA genes, onecould determine whether there was any overlap.This would occur only if a segment of the rRNAgene also coded for a ribosomal protein.

Ribosomal protein pool. When cells are treatedwith CM, or relaxed mutant cells are starved foramino acids, ribonucleoprotein particles (CMparticles or relaxed particles) accumulate whichcontain appreciable amounts of protein [221,238, and other papers reviewed by Osawa (244)].Since on CM treatment no new proteins aresynthesized, most of the protein componentsof CM particles must have been derived frompreexisting proteins (130, 358). Thus, it wasthought that a fairly large pool of ribosomalprotein exists in cells, and its physiological rolein the biosynthesis of rRNA was suggested (155).However, kinetic studies on the incorporationof radioactive amino acids into ribosomal pro-teins in the ribosomes showed that the pool isvery small if it exists at all (96, 279). This dis-crepancy can now be explained in the followingways. (i) At least some of the proteins containedin CM or relaxed particles are nonribosomalproteins absorbed to rRNA nonspecifically(358); therefore, the amount of ribosomal pro-

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tein in the CM particles was over-estimated.(ii) During CM treatment, preexisting ribosomesbreak down and this provides ribosomal proteinsfor the formation of the CM or relaxed particles(163, 238). Thus, although there are some ex-perinrents in which significant amounts of freeribosomal proteins were detected in cell-freeextracts (272) or inferred to be present frompulse-chase experiments (61) and, althoughfree ribosomal proteins certainly exist as a largepool under conditions of ribosome breakdown[e.g., Mg2+ starvation (164, 222)], we are nowcertain that the pool size of free ribosomal pro-teins is small in exponentially growing E. coilcells.

Assembly ProcessNumerous experiments have been performed

to learn the sequence of biochemical eventsoccurring in the ribosome assembly process.Three major approaches have been used: (i)kinetic analysis of the flow of radioactive RNAprecursors into mature ribosomes; (ii) use ofinhibitors to cause the accumulation of inter-mediate particles; and (iii) genetic approaches,that is, use of mutants defective in some stepsin the assembly of ribosomes.

Kinetic analysis of the flow of RNA precursorsinto mature ribosomes. In their pioneering ex-periments, the group at the Carnegie Institution(27) pulse-labeled growing cells with 14C-uracilto observe the flow of label into mature ribo-somes. Because they did not distinguish pulse-labeled unstable mRNA from the ribosomeprecursor RNA, the biosynthetic scheme arrivedat from a kinetic analysis of their data has beenconsiderably modified. Nevertheless, their majorconclusion is still valid: ribosome assembly is astepwise process.More recently, Schlessinger's group eliminated

the problem of mRNA contamination by usinga method of lysing bacterial cells gently. Such amethod allowed separation of the mRNA-containing polysome fraction from the ribosomalprecursors, which are in turn apparently ab-sent from the polysome population (182). Oneimportant question dealt with in their work,which still remains to be answered unam-biguously, is whether rRNA ever exists in a freeform, that is, not associated with any ribosomalproteins. In an earlier study, analysis of pulse-labeled "shift-up" cultures by Kono and Osawa(149) showed two components, sedimenting at17 to 18S and at 22 to 23S, respectively, whichthey claimed to be free rRNA. In contrast to this,Mangiarotti's group (180) claims that ribosomalproteins become attached to the RNA as it isbeing synthesized. They observed the presence

of a whole array of particles containing bothincomplete rRNA chains (sedimenting slowerthan 16S) and proteins. However, they couldnot prove that the proteins complexed with theseincomplete rRNAs are really ribosomal proteins.In any event, they failed to detect free, completedrRNA chains. Instead, they found that thefirst completed chains of 16S and 23S RNA existin the form of ribonucleoprotein particles. Theseparticles are the first kinetic "hold-up" pointsin the synthesis of 30S and 50S subunits. Theyidentified two successive precursors for the 50Ssubunit, sedimenting at 32S and 43S, respectively.[These intermediates had also been identifiedby the Carnegie group (27).] They also identifieda single 30S precursor with a sedimentationcoefficient of 26S. Experiments by Osawa (245,246) are in basic agreement with this scheme,with the exception that they have proposedanother 30S precursor prior to the 26S stage,sedimenting at 22S (see Fig. 4). However, de-detection of 22S particles was only mentioned intheir papers and no experimental details werepublished. Both 22S and 26S particles were saidto contain submethylated precursor 16S RNA["17S" RNA] similar to the "17S" RNA in CMparticles (245). Thus, the RNA in these particlesprobably corresponds to the precursor 16SRNA studied by Adesnik and Levinthal (1).Two precursors for 50S subunits, 325 and 435

particles, have been well characterized (82a,180, 209, 246). These precursor particles contain23S rRNA which is submethylated (about 60%of the normal level) and must correspond tothe precursor 23S RNA studied by Adesnik andLevinthal (1). Osawa's group as well as Morelland Marmur (214) found that the precursorparticles contained less than 30% of the 55RNA found in complete particles. However,43S particles analyzed by Monier and his co-workers (209), which must be very similar to the43S particles analyzed by Osawa's group, con-tained the same amount of 5S RNA as complete50S particles. On subsequent purification, theamount of 5S RNA relative to 23S rRNA de-creased. From these observations, they suggestedthat 435 particles contain 5S RNA but that thebinding of 5S RNA to the particles is weak,possibly because of the absence of some keyproteins or due to the "unfolded" conformationof the 43S particles (209).The protein compositions of both 32S and

43S particles were analyzed by Osawa and hisco-workers (246). Although we now know thatthere are about 35 proteins in the 50S subunit,Osawa's group could resolve them into only 19components after one cycle of column chroma-tography on CM cellulose. Using this chroma-

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[Nascent 16S RNA ("17S" RNA)] -- 21S particle -- 26S particle -. 30S subunit[Nascent 23S RNA] -* 32S particle -* 43S particle -+ 50S subunit

FiG. 4. Biosynthetic pathways of30S and 50S ribosomal subunits in vivo.

tographic method, they found that only 3 of the19 components were present in the 32S pre-cursor particles and an additional 9 of the com-ponents (i.e., a total of 12 of the components)were present in the 43S precursor particles. Theseresults are consistent with the conclusion ob-tained from kinetic studies (180) that the orderof 50S subunit assembly is (nascent 23S rRNA)--+ 32S--4 43S ---+ 50S (see Fig. 4), and showthat addition of proteins to the 23S rRNA issequential.

Ifwe consider these precursors as intermediates,we can ask what factors limit their role of con-version into complete ribosomes. Two possi-bilities can be considered: (i) the synthesis ofsome components, such as 5S RNA or some ofthe ribosomal proteins, is rate limiting; or (ii)some conformational changes of these precursorparticles are necessary for the further additionof proteins. In vitro reconstitution studies aswell as the analysis of cold-sensitive mutantsto be described below support the second view.

Analysis with metabolic inhibitors. The firstexperiment in which a metabolic inhibitor wasused in attacking the problem of ribosomeassembly was performed by Nomura and Watson(238). When growing E. coli B was treated witha high concentration of CM (200 pg/ml), theaccumulation of large amounts of new ribo-nucleoprotein particles (CM particles) containing75% RNA and 25% protein was observed. Theextensive breakdown of preexisting ribosomeswas also found and it was suggested that thisbreakdown provides the proteins to combinewith the newly synthesized rRNA. Subsequentwork showed that CM particles are of two majortypes, 185 and 25S, containing 16S and 23SrRNA, respectively (156). After the discovery ofCM particles, other workers found that a varietyof conditions inhibiting protein synthesis resultedin the accumulation of similar particles: "re-laxed particles" synthesized during amino acidstarvation of relaxed mutant strains (221),"puromycin particles" (56, 130), "streptomycinparticles" (64), and so on. Since at least a majorpart of the rRNA contained in these particleswas converted to the rRNA of mature ribosomesafter conditions allowing protein synthesis wererestored (128, 221, 231), these particles werethought to represent normal intermediateparticles. [For a summary of earlier work sup-porting this view, see the review by Osawa (244)].In fact, several reports claimed that the proteinmoieties in these particles could also be utilized

to form complete ribosomes after protein syn-thesis was resumed (61). However, experimentsby Yoshida and Osawa (358) showed thatparticles similar to CM particles could be arti-ficially produced from nascent rRNA duringthe preparation of cell extracts. A similar con-clusion was reached by Schleif (279). Thus, itnow appears that CM particles do not representnormal intermediate particles. However, manyof the proteins contained in CM particles andrelaxed particles are very similar to genuineribosomal proteins in their gel electrophoreticpattern and are proteins which are not presentin the soluble protein fraction of normal cells(163, 164, 220, 358). It appears that the nascentrRNAs synthesized under conditions of stronginhibition of protein synthesis behave likesynthetic polyanions in vitro (350, 351), in thatthey induce breakdown of preexisting ribosomesand adsorb the released proteins nonspecifically(163). The experiments by Yoshida and Osawajust described (358) indicate that breakdown ofthe ribosomes in normal cells takes place duringthe preparation of extracts from a mixture ofnormal cells and CM-treated cells. The presenceof significant amounts of nonribosomal proteinsin CM particle preparations points to the ne-cessity for caution in the purification of ribosomeprecursor particles as well as of ribosomes.Even ifCM particles are an artifact of extractionthis does not mean that the nascent precursorrRNAs they contain were present in a free forminside the cell before extraction. The experimentsof Goodman et al. (100) mentioned before sug-gest that a few crucial ribosomal proteins alwaysappear with newly synthesized rRNA. Thus, theimportant question whether free rRNA can besynthesized without binding to any ribosomalprotein is still unsettled.

In contrast to the treatment of E. coli cellswith a high concentration of CM, Osawa et al.(246) found that treatment with a low concentra-tion of CM (0.6 to 1.5 gg/ml) caused accumula-tion of 425, 32S, 265, and 22S ribonucleoproteinparticles in addition to mature 50S and 30S par-ticles. They showed that the CM-induced 42Sand 32S particles both contain 23S rRNA andprotein components identical to those of pulse-labeled 42S and 32S particles from normal cells.Thus, these two particles observed in CM-treatedcells appear to represent normal intermediatesin the assembly of 50S subunits. Both 26S and22S components were found to contain the pre-cursor 16S rRNA (17S rRNA). Protein compo-

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nents of these particles were not reported. Judgingfrom the sedimentation properties, CM-induced265 particles may be identical to the 26S particlesobserved by Mangiarotti et al. (180) in short-pulse-labeling experiments. Likewise, CM-in-duced 22S particles may be identical to the 21Sparticles accumulated at low temperatures bysome sad mutants of E. coli which will be dis-cussed in the next section.

Blundell and Wild (20, 21) treated E. coli cellswith 0.3 mM CoCl2, and observed the accumula-tion of at least three particles (44S, 33S, and 23S)in addition-to mature ribosome particles. Thesethree particles may correspond to the 42S, 32S,and 225 particles accumulated in cells treatedwith low concentrations of CM. The reason thesenormal intermediate particles accumulate in thepresence of low concentrations of CM or suitableconcentrations of CoCl2 is not clear. In bothinstances, the inhibitors partially suppress pro-tein synthesis and stimulate RNA synthesis (20,21, 155). The significance of the apparent cor-relation between the unbalanced macromolecularsynthesis and the accumulation of intermediateparticles is unknown.

Finally, it should be mentioned that ribonucleo-protein particles (FU particles) with sedimenta-tion coefficients of 325 and 28S were found inextracts of E. coli treated with 5-fluorouracil (134,149). Although the original claim (149) that theseparticles were converted directly to mature 50Sand 30S subunits was not confirmed and it wasfound instead that these particles break downafter the removal of fluorouracil (7, 125), theymay correspond to the 325 and 26S intermediatesdetected in pulse-labeling experiments. The RNAsin the 325 and 285 FU particles were 23S and165 rRNA, but contained large amounts offluorouracil instead of uracil. It is possible thatreplacement of uracil with fluorouracil causesstructural alteration of the 325 and 26S particlesand prevents conversion of these particles to 40Sparticles and 30S subunits, respectively, presum-ably by preventing binding of the next group ofproteins. It should be informative to analyze theprotein compositions of 32S and 285 FU par-ticles and to compare them with those of the 32Sand 26S particles detected in pulse-labeling ex-periments.The formation of ribosomes in vivo was studied

under several other abnormal conditions. Forexample, Mg+2-starvation in E. coli cells leads todisappearance of ribosomes (164, 189, 222). Ad-dition of Mg+2 to such cells causes the preferentialsynthesis of rRNA, and the newly synthesizedrRNA is usually found in incomplete particlessimilar to CM particles or relaxed particles (222).However, recent experiments by Lefkovits and

DiGirolamo (164) showed that complete mature305 and 50S subunits do form under certain con-ditions and that their formation is insensitive toCM. When they used rich media containing CMduring recovery from Mg+2 starvation, incom-plete particles rather than mature ribosomes wereproduced. Lefkovits and DiGirolamo (164)showed the presence of many basic proteinssimilar to ribosomal proteins in extracts of Mg+2-starved cells, but not in extracts of normal cells.Although these workers have not shown thatribosomal particles formed during recovery inthe presence of CM are biologically active, theirdata strongly indicate that the breakdown ofribosomes by Mg+2 deprivation produces all theribosomal proteins in the pool. Complete riboso-mal particles are produced utilizing these pre-existing ribosomal proteins and newly synthesizedrRNA even in the absence of new protein synthe-sis. Their data also indicate that the overproduc-tion of rRNA in a rich medium without thefurther supply of ribosomal proteins inhibits theformation of complete ribosomes and particlessimilar to CM particles (25S and 18S CM par-ticles) are produced (164, 222). It should be re-called in this connection that, when the ratio oftotal 30S ribosomal proteins to 16S rRNA is al-tered in the in vitro 30S reconstitution system,efficient reconstitution of ribosomes was observeddown to a protein-RNA ratio of about 0.5 (inmoles), indicating a cooperativity in the assemblyin vitro. However, when the ratio was decreasedto 0.2 (in moles), the efficiency of the assemblyreaction was greatly reduced (237). Thus, it ap-pears that the cooperative assembly of ribosomestakes place only within a certain range of ribo-somal protein-rRNA ratios and the cooperativitybreaks down both in vitro and in vivo when therelative amount of rRNA becomes too high.However, the cooperative nature of the assemblyreaction is still biologically advantageous since itdoes not require a very strict coordination of thesynthesis of rRNA and ribosomal proteins. As-sembly takes place even under conditions in whichrRNA and ribosomal proteins are producedseparately without coordination, as revealed bythe experiment of Lefkovits and DiGirolamo(164).In connection with the in vivo self-assembly

process discussed above, it is appropriate to notethe work done by Sparling and his co-workers(291a), using diploid strains of E. coil doublyheterozygous for both str and spc loci. Doubleheterozygotes with the two resistance alleles cis(spcrstrr/spct str) or those with trans (spcrstr/spc' strr) formed essentially identicalsets of ribosomes, as judged by their degreeof resistance to either drug or to both drugs

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together. Thus, it can be concluded that the pro-teins controlled by these genes (P4 and Plo, re-spectively) are assembled into ribosomes from acommon pool rather than by preferential aggrega-tion of products from the same genome (or thesame "operon").Ribosome assembly defective mutants. Genetics

has been a powerful tool in identifying the flow ofintermediates in numerous biosynthetic pathwaysas well as for obtaining information on the mech-anism of their regulation. Thus, it should also beuseful for analysis of the mechanism of ribosomeassembly in vivo. There are now several mutantsknown that have some alteration in the biosynthe-sis of ribosomes.Lewandowski and Brownstein (168) observed

an accumulation of 43S ribonucleoprotein in ad-dition to 50S and 30S ribosomal subunits in amutant which was isolated as a suppressed "re-vertant" from a Sm-dependent E. coil strain. Thenumber of 43S particles relative to the number of50S and 30S subunits in growing cells was fairlylow. Pulse chase experiments showed that the 43Sparticles are a precursor for 50S subunits. The43S particles contain 23S rRNA and many of the50S ribosomal proteins (169). Since the auto-radiographic pattern of the radioactive proteinsin the 43S particles was compared only with thedye staining pattern of the proteins from 50Sparticles on polyacrylamide gel electrophoresisand since the resolution of components was notvery high, it is difficult to conclude whether thedifferences in the protein compositions of 43Sand 50S particles are real, and if so, how manyproteins are missing in 43S particles. However,judging from the published data, the difference,if any, between the protein compositions of 50Sand 43S particles is small; therefore, the accumu-lated 43S particles appear to be different from the43S particles analyzed by Osawa and his co-workers in which 7 of the 19 protein peaks nor-mally found in their column chromatogram for50S particles were missing.Although Lewandowski and Brownstein (168)

have not proved that the accumulation of 43Sparticles is a direct result of the mutation suppres-sing streptomycin dependence, this is a likelypossibility. They have shown that ribosomes fromthis dependence-suppressed mutant pare sensitiveto Sm in in vitro polypeptide synthesis (32). Thusthe suppressor appears to affect a ribosomal com-ponent. Although they have not reported subunitlocalization of the altered component, studies byother workers suggest that most suppressors ofSm dependence cause alterations within the 30Sribosomal subunit (9a; Guthrie, Nashimoto, andNomura, unpublished data; Kurland and Birge,personal communication). Thus, there is a good

possibility that the observed accumulation of 43Sis a result of a structural alteration of a 30Sribosomal component.Another mutant which accumulates a large

number of 43S particles during exponentialgrowth was isolated by MacDonald, Turnock,and Forchhammer (179). This mutant has about80% more RNA per milligram of protein than theparent strain, and this permitted its isolation by abuoyant density method (178). The growth ratewas slower than that of the parent by a factor of2 to 3, and the 70S ribosomes were less activethan those of the parent; this property was cor-related with the slow growth of the mutant. Itwas suggested that a single mutation affecting oneof the components of the 50S subunit leads to thealteration in both the synthesis and function ofthe ribosomes (179). However, subsequent geneticanalysis (342) showed that the complex phenotypeof the mutant is due to the presence of at leasttwo mutations, both of which are closely linkedto the xyl locus. One of the mutations is ap-parently responsible for the accumulation of the43S particles, whereas the other mutation is as-sociated with the reduced growth rate and analtered degree of resistance to Sm that was ob-served subsequently as another feature of thismutant. At the moment, there is no experimentalinformation about the biochemical nature of thedefect which causes the accumulation of 43Sparticles.

In the two mutants accumulating 43S particlesdescribed above, mature ribosomal subunits werealways produced; that is, the block in the bio-synthesis of 50S subunits was not complete. Thusthe conditions for precursor accumulation andconversion were not well defined. There existedno systematic method of isolating mutants withcomplete blocks in ribosome assembly. Sincesuch blocks would be lethal to the mutant cell,such mutants could be isolated only as conditionallethal mutants. Both Ingraham's group (320) andour own (110, 111) found independently that suchmutants could be isolated as cold-sensitive mu-tants.As described in a previous section, one remark-

able feature of the in vitro assembly of 30S sub-units is the high dependence of the rate of as-sembly on temperature. The assembly reactionwas extremely slow at 10 C or below, at whichtemperatures intermediate particles sedimentingat about 21S accumulated. It was concluded thata high activation energy is necessary for thestructural rearrangement of the intermediate. Itwas then reasoned that many mutations affectingthe assembly process in vivo should manifestthemselves more clearly at lower temperatures.In fact, when cold-sensitive mutants of E. coll

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were isolated in a complex medium and examinedfor their ability to assemble ribosomes, a significantfraction of these mutants was found to be defec-tive in some steps of ribosome subunit assembly at20 C (110). These mutants were called ribosomesubunit assembly defective, or sad.Three classes of sad mutants were found and

studied in our laboratory (110): (i) mutants whichfailed to synthesize 50S subunits and accumulated32S particles at 20 C, (ii) mutants which failed tosynthesize 50S subunits and accumulated 43Sparticles at 20 C, and (iii) mutants which showeddrastic defects in both 50S and 30S subunits as-sembly and accumulated both 32S and 21S par-ticles at 20 C. Four mutants were studied in de-tail: sad-19, representing the first class; sad-68,representing the second class; and sad-38 andsad-410, both of which represent the third class.Both the 43S particles accumulated by sad 68

and the 32S particles accumulated by sad 19 at20 C were found to contain 23S rRNA. Both ofthese particles labeled at 20 C were quantitativelyconverted to 50S subunits upon temperatureshift-up to 42 C. Thus, both 32S and 43S par-ticles appear to be precursors of 50S subunits.Because of some mutational defects, conversionof 32S to 43S is blocked at 20 C in the mutantsad 19, and conversion of 43S to 50S is blockedat 20 C in the mutant sad 68. The cold sensitivityof these mutants was shown to be due to a defect.in assembly, and the ribosomes isolated fromthese mutants grown at 40 C were not themselvesfunctionally cold sensitive. The sad-19 mutationcausing 32S particle accumulation was mappedat a locus very closely linked to the spc-locus,which determines the 30S ribosomal protein P4.Genetic analysis showed that cold sensitivity, theinability to synthesize 50S subunits, and the ac-cumulation of 32S particles at 20 C all appear tobe due to a single mutation at this locus. Thelocation of the sad-19 locus at the spc-str regionindicates one of two possibilities. (i) A 50Sribasomal component or some extra ribosomalcomponent is the one altered, and the sad-19locus is linked to the genes controlling 30S ribo-somal proteins, or (ii) a 30S ribosomal componentis the one altered by the sad-19 mutation, and theassembly of the 50S subunit is somehow depend-ent on the correct structure of the 30S subunit orits precursor. The genetic analyses of drug resist-ance mutations in B. subtilis support the formerpossibility (99, 288), whereas esperiments onsad 38 discussed below suggest the latter.The genetics of the sad-68 mutation, which

causes accumulation of 43S particles, is not clearat this time. It is also not clear whether the 43Sparticles accumulated in this mutant are the sameas the 43S particles of MacDonald et al. (179) or

Lewandoski and Brownstein (168), or thosestudied by Osawa and his co-workers (246). Pre-liminary experiments in our laboratory (Nashi-moto, unpublished data) indicated that the 43Sparticles accumulated by sad-68 appear to containmost of the 50S proteins. Precise comparisonwith the protein composition of the wild-type 43Sintermediate detected by pulse labeling has notbeen done.The sad-38 and sad-410 mutants which accumu-

lated both 21S and 32S particles at 20 C werestudied in detail. The 32S particle contains 23SrRNA and appears to be a precursor of the 50Ssubunit. The 21S particle contains "17S" rRNA,which can be distinguished from mature 16SrRNA by polyacrylamide gel electrophoresis aswell as by sucrose density gradient centrifugation.The protein composition of 21S particles wasstudied by column chromatography as well as bypolyacrylamide gel electrophoresis. The particleswere found to contain nine of the 30S proteins.No significant amount of protein was found thatwas not 30S ribosomal protein. Some mutationaldefect apparently prevents attachment of some ofthe 30S ribosomal proteins while allowing thebinding of others to the "17S" rRNA to formthe 21S particles. The protein composition of 21Sparticles is important for checking the validity ofthe order of addition of 30S proteins to 16SrRNA as determined in vitro. The following pro-teins were definitely identified in the 21S particles(Nashimoto and Nomura, unpublished data): P4a,P4b, P5, P8, P9, Plo0, Pi3, and PI4. One more proteinpresent in the particles is not definitely identifiedand could be P3a, P3b, or P30. These proteins areall proteins involved in the early steps of theribosome assembly process according to the invitro scheme (see Fig. 3). Thus the consistency ofthe in vivo data with the in vitro scheme is verystriking. The protein composition of these 21Sparticles is also similar to the protein compositionof 21S RI particles obtained in the in vitro re-construction system.A genetic analysis of the sad-38 mutation (111)

revealed that the mutation is closely linked toaroE, but appears to be located on the oppositeside of aroE relative to the spc-str region. Againthe cold-sensitive phenotype, defective 50S bio-synthesis and the accumulation of 21S particlesat 20 C appeared to be due to a single mutation.The apparent simultaneous defects in both 50Sand 30S subunit assembly suggest that the bio-synthesis of 30S subunits is somehow coupledwith that of 50S subunits. A single mutation ina 30S ribosomal component may affect both 30Sand 50S subunit assembly. It is interesting to notein this connection that a significant fraction ofspontaneous Sm-independent "revertants" de-

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rived from a Sm-dependent strain is cold sensitiveand accumulates both 30S and 50S precursors at20 C (Nashimoto and Nomura, unpublished data).Detailed studies of this pehnomenon may reveal apossible mechanism which insures the coordinatedassembly of 30S and 50S subunits in vivo.

Using Salmonella typhimurium, Tai and his co-workers also found that a significant fraction ofthe cold-sensitive mutants isolated in a complexmedium has abnormal ribosomal biosynthesis atlow temperatures (320). They studied two mu-tants, PT11 and PT117, in detail. The mutationsinvolved were shown to be linked to the str locusand to cause overproduction of RNA relative toprotein at 20 C. Thus, the RNA-to-protein ratioin the mutant cells was the same as in the parentstrain at 37 C, but it was significantly higher thanthat in the parent at 20 C. Sucrose gradient sedi-mentation analysis of ribonucleoprotein particlessynthesized by the mutant PT1 17 at 20 C failed toreveal any accumulation of intermediate particles,but indicated an increased amount of RNA atthe 30S region. However, the published datacannot exclude a possible accumulation of par-ticles at both the 26S and the 325 regions, leadingto the apparent overproduction of "305" par-ticles. In contrast the other mutant, PT11, wasshown to accumulate a ribonucleoprotein particlein the 22S region. These particles appear to beanalogous to the 21S particles accumulated insad-38 and sad-410 mutants at 20 C.A priori, there are several possible causes of

defects in ribosomal assembly leading to the ac-cumulation of intermediates. (i) The rRNA isaltered and cannot bind some of the ribosomalproteins properly. (ii) A ribosomal protein isaltered, and the altered protein fails to bind to anintermediate particle. (iii) A ribosomal protein isaltered, and an intermediate particle fails to bindadditional components because of some structuraldefect caused by the altered protein in that inter-mediate. (iv) Some enzymes which modify ribo-somal components (e.g., rRNA methylase) aredefective, leading to the accumulation of defectiveribosome intermediates. (v) Mutations are insome hypothetical nonribosomal componentsparticipating in ribosome assembly.

In view of the availability of many useful mu-tants as well as developments in the techniques ofribosome reconstitution and protein fractionationwhich can be used for identifying mutationalalterations, it appears that the genetic approachwill prove to be very useful in analyzing the mech-anism of the ribosomal assembly process in vivo.

CONCLUDING REMARKSThe study of complex cellular organelles such

as ribosomes has appeared to be a formidable task.

However, recent developments in the determina-tion of the structure, function, and assembly ofribosomes, especially of 30S subunits, describedin this review make us optimistic about the furtherstudy of these particles. Instead of a somewhatspeculative description of the ribosome, a blackbox in the discussion of protein synthesis in thepast, we now have much solid experimental in-formation. Although the complete three-dimen-sional structure of ribosomes cannot yet bedescribed, it seems that a first approximation tothis goal may be achieved in the near future, atleast with respect to the 30S subunit. Knowledgeabout the regulatory mechanisms controlling thebiosynthesis of a complex organelle such as theribosome will be important to our understandingof development and morphogenesis in general.

Thus, the rapid, recent progress in the study ofthe molecular organization of ribosomal particlesshould not be regarded as the approach to theend of our research but rather as a beginning stepin the study of the biogenesis of other compli-cated, yet specific, biological-structures.

ACKNOWLEDGMENTS

This investigation was supported by Public Health Servicegrant GM 15422 from the National Institute of General Med-ical Sciences and by grants from the National Institutes ofHealth and the National Science Foundation.

I thank Maria Brzezinska and Christine Guthrie for their helpin preparation of the manuscript and Julian Davies for his usefulcomments.

ADDENDUM IN PROOFAlthough experiments performed by Nomura et al.

(234) failed to detect any functional contribution forprotein P1, Kurland and his co-workers subsequentlyfound stimulatory activity of this protein in poly Ubinding as well as poly U-dependent phe-tRNA bind-ing (personal communication). In the experiments byNomura et al., particles obtained after reconstitutionwere washed in high-ionic-strength buffers. Underthese conditions, binding of P1 to the reconstitutedparticles was very weak (201). This may explain whyno functional difference was observed between recon-stituted particles made in the presence of P1 andthose made in the absence of P1.

LITERATURE CITED

1. Adesnik, M., and C. Levinthal. 1969. Synthesis and matura-tion of ribosomal RNA in Escherichia coli. J. Mol. Biol.46:281-303.

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