Vol. 152, No. 2JOURNAL OF BACTERIOLOGY, Nov. 1982, P. 661-6680021-9193/82/110661-08$02.00/0Copyright C 1982, American Society for Microbiology

Escherichia coli Phenylalanyl-tRNA Synthetase Operon:Transcription Studies of Wild-Type and Mutated Operons on

Multicopy PlasmidsJACQUELINE A. PLUMBRIDGE* AND MATHIAS SPRINGER

Institut de Biologie Physico-Chimique, 7S005 Paris, France

Received 2 April 1982/Accepted 26 July 1982

The construction of three X bacteriophages containing parts of the structuralgene for threonyl-tRNA synthetase, thrS, and those for the two subunits ofphenylalanyl-tRNA synthetases, pheS and pheT, is described. These phages wereused as hybridization probes to measure the in vivo levels of mRNA specific tothese three genes. Plasmid pBl carries the three genes thrS, pheS, and pheT, andstrains carrying the plasmid show enhanced levels of mRNA corresponding tothese genes. Although the steady-state levels of threonyl-tRNA synthetase andphenylalanyl-tRNA synthetase produced by the presence of the plasmid differedby a factor of 10, their pulse-labeled mRNA levels were about the same. Mutantderivatives of pBl were also analyzed. Firstly, a cis-acting insertion locatedbefore the structural genes for phenylalanyl-tRNA synthetase caused a majordecrease in both pheS and pheT mRNA. Secondly, mutations affecting eitherstructural gene pheS or pheT caused a reduction in the mRNA levels for bothpheS and pheT. This observation suggests that autoregulation plays a role in theexpression of phenylalanyl-tRNA synthetase.

The synthesis of proteins is one of the mostcomplicated processes carried out by the bacte-rial cell. It involves many different macromol-ecules and macromolecular complexes, e.g.,ribosomes, tRNAs, aminoacyl-tRNA synthe-tases, initiation, elongation, and termination fac-tors, and various proteins involved in matura-tion. The coordinated synthesis and functioningof all of these compounds evidently necessitateeffective regulation to produce the balancedgrowth of the cell. The balanced growth of thecell also requires that the component metabo-lites, in particular the amino acids, are synthe-sized in a controlled manner. The biosynthesisof amino acids is modulated by both the externalconditions, i.e., whether a particular amino acidis present or not in the growth medium, andinternal consumption at the level of proteinsynthesis.Aminoacyl-tRNA synthetases and tRNAs are

the link between the translation machinery andamino acid metabolism. These two classes ofmacromolecules assume a central role both inthe mechanism of translation and in the regula-tion of the expression of amino acid biosyntheticenzymes (22). It is thus a matter of considerableinterest to know how these macromolecules arethemselves regulated.

Several aminoacyl-tRNA synthetases havebeen shown to be subject to metabolic regula-

tion, i.e., their synthesis increases with increas-ing growth rates (8). The aminoacyl-tRNAsynthetases do not seem to be affected by thestringent response in a coordinate manner: someshowed a moderate stringent response, whereasothers seemed unaffected (2). An increase inaminoacyl-tRNA synthetase levels has been ob-served upon amino acid limitation in a certainnumber of cases (10). Phenylalanyl-tRNA syn-thetase, in particular, showed a long-term dere-pression upon phenylalanine starvation in a bra-dytroph (7).We have been studying the regulation of the

expression of the genes for two aminoacyl-tRNA synthetases, those for threonine (thrS)and phenylalanine (pheS and phe7), whosegenes are grouped at 38 min on the Escherichiacoli chromosome (3, 16, 17). These genes wereinitially isolated on a X transducing phage, Xp2(17), and hence, subcloned on multicopy plas-mid pBl (13). The direction of transcription andthe number of transcription units in the regionhave been determined (11, 18). Several sponta-neous mutants of plasmid pB1 which exhibitaberrant protein synthesis patterns are charac-terized in the accompanying paper (12). Tofurther study the expression of the genes onpBl, we constructed three A transducing phagescontaining DNA from within the structuralgenes thrS, pheS, and pheT. We used these

661

on June 14, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

662 PLUMBRIDGE AND SPRINGER

phages as gene-specific hybridization probes tomeasure the levels of mRNA from both wild-type and mutant plasmids.

MATERIALS AND METHODSStrains and general methods. The E. coli and bacte-

riophage A strains and the plasmids used are listed inTable 1. The structure of pBl is shown in Fig. 1. pBland the mutated plasmids derived from pBl, pBlS andpBlT, were described in the accompanying paper (12).pBlS carries a 1.3-kilobase (kb) insertion before thestructural genes pheS,T of phenylalanyl-tRNA synthe-tase within the fragment HpaI5-AvaI3. This insertionhas been designated as fl(pheSp::1.3 kb)1001. pBlT isa 20- to 30-base-pair (bp) deletion between AvaI3-PstI3, i.e., within the structural gene pheS. Thisdeletion has been designated ApheSO002. pB21 is anin-vitro-constructed deletion of the BamHI1-BamHI2fragment of pBl. This deletion removes the COOH-terminal part of pheT and the beginning of the genesconferring resistance to tetracycline (19). pB21 still

complements pheS mutations and confers resistanceto ampicillin.

General genetic methods were as described else-where (6, 16). Recombinant DNA techniques were asdescribed previously (13) or as described in reference4. The construction of the gene-specific phage probesfor thrS, pheS, and pheT is shown in Fig. 1 anddescribed below.

Large-scale purification of phage A DNA. PhagesXgt4 and Xp2 were grown by the "preabsorbtion,dilute, shake" method (1). The phage particles wereprecipitated with polyethylene glycol 6000 (21). Phagescarrying the S7 mutation, as well as thermosensitiverepressors (XM100, XpU6, XpS5, and XpT3), weregrown by the thermoinduction of lysogens in bacteriawhich do not carry a suppressor for the S7 ambermutation. The induced bacteria were collected bycentrifugation and lysed with chloroform. Bacterialhost DNA was digested with 10 p,g of DNase per ml for30 min at 37°C, and cellular debris was removed bycentrifugation. Phages were purified by two succes-sive cycles of CsCl stepwise gradient centrifugation,

seeal ski P.XI.Bb (I Rg)

h(A)

X65.

P680 Xh69.O>6K i21ts

CI857 lb5 Agt4

X825 S83.4E A A2 A 3 Hi 02

i (1) X65.6 Xh169 i.m20ts-:.1 .:I: :-

Im

1tI:AA

1(A) X65.6 A8l i.21t(1) I m2lt3

h (A) C1351 z .aD4 o .lI ApT3..;.. . .._

S3 815% A 1kb

FIG. 1. Construction of gene-specific phage probes. The right-hand arm of the two starting phages Xgt4 andKM100 is shown with the relevant restriction sites. The position of the sites is given in percent units taken fromreference 20 or estimated from reference 15 for the PstI sites. Symbols: =, imm2lts substitution; _, ninSdeletion; _, thrS; =, inJC; _, pheT; m, pheS; the expected structures of the recombinant phages areshown underneath. 57 and c1857 indicate the position of these mutations in XM100 and Xgt4, respectively.Plasmid pBl is shown on a somewhat larger scale with relevant restriction sites and the positions of thecharacterized genes. Only those restriction sites used in the cloning are given. XpU6, thrS probe; XpS5,pheSprobe; XpT3, pheT probe. Restriction sites designations: E = EcoRI, P = PstI, A = AvaI, S = SacII, B =

BamHI, Hd = HindIII, Xh = XhoI, X = XmaI ( SmaI). Site AvaIl ofpB1 is a XhoI site. Sites AvaI2 and AvaI3of pBl are XmaI sites.

B861S7 AMIoo

EpBl

S7 ApU6

Si ApS5

II )IW WWI I I . v 16W I %F .m.. I.I. I.,. I. I.

I I- 'imi

thr s ;k p ph

J. BACTERIOL.

=:,

08#1 A DOLE I

on June 14, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

E. COLI PHENYLALANYL-tRNA SYNTHETASE OPERON 663

TABLE 1. E. coli and bacteriophage A strains used

Strain Relevant genotypes' Source orreference

LE392 F- hsdR hsdM+supF R. DavissupE44 (leu, thi, thr,thy)?

IBPC1365 F- thi-l argE3 his4 (18)proA2 lacY) galK2mtl-l xyl-5 tsx-29supE44 A-

IBPC2001 recAl rpoB, other (18)markers as IBPC1365

IBPC4501 aroD6 rpsL, other (18)markers as IBPC1365

PhageXM100 h(480) imm2l clts S7 M. HoffnungXgt4 A [EcoRl (40o-Lac)- R. Davis

EcoRI (54.3%)] c1857nin5

Xp2 thrS infC pheS pheT (17)d1857

ApT3 'pheT' c1857 S7 This workApS5 pheS' imm2l clts 57 This workXpU6 'thrS' imm2l clts S7 This work

a ', Indicates that the gene is incomplete on thatside.

first descending and then ascending as described else-where (4). Phage DNA was isolated either by phenolextraction or by formamide treatment (4). Single-stranded phage DNA was prepared as described byMiller (6) and dialyzed into 2x SSC (SSC is 15 mMsodium citrate, 150 mM NaCI).Southern blotting and hybridization. Digested DNA

fragments were analyzed by 1% agarose gel electro-phoresis in Tris-acetate buffer. Southern transfer wascaried out as described elsewhere (4), using 20x SSCas eluant for 12 to 18 h. After washing and drying invacuo at 80°C, hybridization was carried out in 5xSSC with 100 ,ug oftRNA per ml as the carrier for 20 h

at 65°C, using 3.4 x 106 cpm of [3H]uridine-pulse-labeled mRNA prepared from IBPC136S(pBl). Thewashed and dried filter was treated with En3Hancespray (New England Nuclear Corp., Boston, Mass.)and exposed to RP X-Omat film at -70°C for 1 week.

Labeling and extraction of mRNA. IBPC2001 wasused to host pBl and the mutant plasmids. Cells weregrown in the synthetic morpholinepropanesulfonicacid (MOPS) medium of Neidhardt et al. (9) supple-mented with either the amino acids specifically re-quired by the strain (arginine, histidine, and proline at50 ,ug ml-') or with all of the amino acids (8) and theappropriate antibiotics (50 Fg of ampicillin per ml and10 ,ug of tetracycline per ml). Cytidine and uridine (50pg ml-') were also included. At an absorbance at 650nm (Awo) = 0.4, the cells were washed free of thepyrimidines and suspended in fresh medium minusuridine and cytidine; labeling with [5-3H]uridine wasgenerally for 2 min with 100 ,uCi ml-' (AmershamInternational plc. Amersham, U.K.; specific activity,30 Ci mmol-'). Cells were immediately lysed by beingpipetted into a 1/10 volume of boiling sodium dodecylsulfate solution, and mRNA was extracted with phenoland CHC13 in the presence of 0.2 M sodium acetate(pH 5.2) as described elsewhere (5, 11). After ethanolprecipitation, any contaminating plasmid DNA wasremoved by digestion with iodoacetate-treated DNase(11, 23). After a second phenol-CHCl3 extraction, themRNA was precipitated with ethanol and finallystored in sterile water at -20°C.Hybridization between single-stranded ADNAs and

[3HJurkdine-labeled mRNA. Hybridization was carriedout in 2x SSC at 65°C for 5 h. After treatment with 10"g of RNase per ml (heated at 100°C for 2 min),hybrids were collected by filtration on HAWP 0.45-,unfilters (Millipore SA, Molsheim, France) and washedwith 40 ml of 10 mM Tris (pH 7.4)-150 mM KCI.Finally, the filters were washed with 5 ml of 70%oethanol before being dried and counted. The input ofradioactive mRNA was 2.5 x 104 to 6.0 x 104 cpm.The saturating amount of single-stranded DNA wasdetermined empirically for each batch; in general,about 0.5 to 1 ,ug of separated strand was used with

TABLE 2. Relative levels of pheS, pheT, and thrS mRNA in IBPC2001 carrying various plasmids

Growth Labeling Bi-specific mRNA Molar ratios of gene-specific mRNAPlasmid condition time (min) (% input)a pheT pheS thrS

pBl A 2 28.5 1.39 1.76 1A 1 30.5 1.35 1.49 1B 2 13.2 0.61 0.73 1B 2 10.2 0.71 1.02 1B 10 7.7 1.07 1.87 1

pBlS A 2 14.7 0.1 0.14 1B 2 8.6 0.08 0.11 1

pBlT A 2 16.5 0.38 0.49 1B 2 17.2 0.22 0.27 1

pB21 A 1 11.5 0.4 1

a The percent Bl-specific mRNA is calculated as the percentage of input counts per minute hybridizing to theheavy strand of Xp2 (i.e., total transcripts from the Bi region). For the specific gene probes, the countshybridizing to each A were corrected for the size of the inserted gene-specific DNA giving counts per minutehybridized per kb of DNA. These values were then normalized to the thrS value. Growth condition A is theMOPS minimal medium (9) supplemented with only auxotrophic amino acids, arginine, histidine, and proline.Growth condition B is the MOPS medium supplemented with all of the amino acids (8). Two independentpreparations ofpBl mRNA under both conditions were analyzed and indicate the reproducibility of the system.

VOL. 152, 1982

on June 14, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

664 PLUMBRIDGE AND SPRINGER

mRNA derived from 0.015 A650 U of cells. On theorder of 1 to 3% of the input counts hybridized to thegene-specific probes. The data reported in Table 2 arethe result of triplicate measurements at two differentconcentrations of single-stranded DNA with a fixedamount of labeled mRNA. Background values (countsretained on the filter in the absence of single-strandedDNA) were low and normally less than 10% of thecounts retained in the presence of the gene-specific Xprobe DNAs.

RESULTSConstruction and characterization of K probes

for thrS, pheS, and pheT. (i) Cloning of gene-specific fragments in X. An asymmetric cloningprocedure was adopted to insert into X phagesfragments of pBl which are specific for thestructural parts of the thrS, pheS, and pheTgenes. The fragments we wished to clone werefor thrS, AvaIl-AvaI2 (AvaIl is a XhoI site, andAvaI2 is an XmaI site); for pheS, AvaI3-PstI3(A-vaI3 is an XmaI site); and for pheT, BamHIl-SacII2 (Fig. 1). The principle of the method forcloning a fragment located between sites "X"and "Y" was to use the left arm of one X endingwith a site X and the right arm of a second Xending with a site Y and then ligate into this pBlDNA digested with both X and Y enzymes.The two phages we chose to use were XM100,

a host range 080, imm2lcItsS7 phage and Xgt4(4), a derivative of XplacS, deleted betweenEcoRI sites at 40% (in lac) and 54.3% of X' andwhich is cI857 and ninS (Fig. 1).To construct the thrS probe, Xgt4 was digest-

ed with XmaI, giving, among others, the 0 to65.6% fragment, and XM100 was digested withXhoI, giving the 69.0 to 100% fragment, withothers derived from the 4)80 part of XM100 (Fig.1). These digested phages were ligated with pBldigested with XhoI and XmaI and then used totransfect LE392. The resultant phages werescreened for host range X, imm2l, and S7 char-acters. The pheS probe was constructed to con-tain the left-hand arm of Xgt4 up to the XmaI(65.6%) site and the right-hand arm of XM100from the PstI (68%) site with the AvaI3-PstI3fragment of pBl (Fig. 1). After the digestion ofXgt4 with XmaI, XM100 with PstI, and pBl withboth enzymes, the DNAs were mixed, ligated,and used to transfect LE392. The resultingphages were screened for host range X, imm2l,and S7 characters. The pheT probe was con-structed to contain the left-hand arm of Xgt4 upto SacIl (83.4%) and the right-hand arm ofXM100 from the BamHI site (86.1%) with theBamHIl-SacII3 fragment of pBl (Fig. 1). Afterdigestion of Xgt4 with SacIl, XM100 withBamHI, and pBl with both enzymes, the mixedligated DNA was used to transfect LE392. Theresulting phages were screened for host range X,c1857 (X immunity), and S7 characters. Starting

with 1.5 ,ug of each DNA and 1 ,ug of pBl DNA,we isolated and purified 5 potential pheS probes,4pheTprobes, and 11 thrS probes. We expectedto obtain phages with the structures indicated inFig. 1 for XpT3, XpS5, and XpU6 as the pheT,pheS, and thrS probes, respectively.

(ii) Analysis of the DNA structure of recombi-nant phages. Lysogens of all of the recombinantphages were made with IBPC1365 or IBPC4501.The phages all carry thermosensitive repressorsand are S7. Thus, high-titer lysates were ob-tained from small cultures after thermoinduc-tion. DNA was obtained from these lysates bythe rapid method described by Davis et al. (4).The analysis of the recombinants with variousrestriction enzymes identified the required bandof pBl, but in all cases, there was additionalDNA which could not be assigned to either thestarting X's or pBl. The phages were all differ-ent; therefore, we took for each probe the phagecontaining the requisite pBl fragment with theleast amount of extraneous DNA and analyzed itfurther. We estimate that XpT3 (the pheT probe)contains an additional 1.4 kb, XpS5 (pheS probe)contains an extra 0.8 kb, and XpU6 (thrS probe)contains an extra 3 kb. The origin of this addi-tional DNA is unknown, but we suspect it arosefrom in vivo recombination during the transfec-tion step since it is of varying sizes and notdefined by any of the restriction enzymes usedin the cloning. We could not eliminate the possi-bility that it derived from pBl. Because addi-tional pBl DNA could invalidate our measure-ments of mRNA specific to one gene carried bypBl, we analyzed our three probe phages bySouthern blotting and mRNA/DNA hybridiza-tion.

(iii) Characterization of the phage probes bySouthern blotting and mRNA hybridization. Thepurified phage and pBl DNAs were digestedwith the relevant pairs of enzymes used in thecloning, and their products were analyzed byagarose gel electrophoresis (Fig. 2A). Southernblotting was carried out as described above.Pulse-labeled mRNA isolated from IBPC1365(pB1) was hybridized to the blot whose fluoro-gram is shown in Fig. 2B and 2C. The only bandof XpS5 digested with SmaI and PstI that hybrid-izes to mRNA extracted from IBPC1365(pBl)corresponds to the AvaI3-PstI3 fragment of pBl(Fig. 2, lanes 4 and 5). Similarly, the only bandof XpT3 digested with SacIl and BamHI thathybridizes to the same mRNA corresponds tothe BamHIl-SacII3 fragment of pBl (Fig. 2,lanes 2 and 3), and the only band of XpU6digested with XhoI and SmaI that hybridizescorresponds to the AvaIl-AvaI2 fragment ofpBl(Fig. 2, lanes 6 and 7). Extending the exposureof the fluorogram did not reveal any additionalbands hybridizing to pBl-specific mRNA.

J. BACTERIOL.

on June 14, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

E. COLI PHENYLALANYL-tRNA SYNTHETASE OPERON 665

A l 2 3 4 5 6 1 81 2 3 4 5 6 7

.Xa

1t....

_

....

-A3-P3 -*

O-A2-A2

A=AvaIP Pst IB= BamHIS Sacd

c 123K 45 7

AP3-_A1 A2 .:X'.+'..

..4..

FIG. 2. Southern blot analysis of the gene-specific phage probes. (A) Agarose gel electrophoresis (1%) ofXpT3, XpS5, XpU6, and pBl DNA digested with pairs of enzymes used in the construction of the phage probes.DNA is visualized by ethidium bromide staining. (B) Fluorogram of the same gel after Southern blotting andhybridization with IBPC1365(pB1) mRNA. Exposure was for 1 week. (C) As in (B) but with a 2-month exposureof the fluorogram. Only the lower part of the fluorogram is shown. Lane 1, pB1 digested with PstI; lane 2, pBldigested with BamHI and SacJ; lane 3, XpT3 digested with BamHI and SacII; lane 4, pB1 digested with PstI andSmaI; lane 5, XpS5 digested with PstI and SmaI; lane 6, pBl digested with XhoI and SmaI; lane 7, XpU6 digestedwith XhoI and SmaI. The arrows indicate the common bands which hybridize between pBl and the phageprobes. Lanes 2 and 3, BamHIl-SacII3; lanes 4 and 5, AvaI3-PstI3; lanes 6 and 7, AvaIl-AvaI2. Each pB1 lanecontains about 1 ,ug of DNA, and each phage probe lane contains about 3 ,ug of DNA. (All XhoI and SmaI sitesare also AvaI sites, and the recognition sequences for SmaI and XmaI are identical.)

(iv) Orientation of the inserted pBl fragmentsin the phage probes. The two strands of the threeprobes (ApS5, XpT3, and XpU6) and of Xp2 (17),which carries all of the genes of pBl (13), wereseparated as described elsewhere (11). Bothstrands of each phage were tested for hybridiza-tion with labeled mRNA made from pBl-carry-ing cells. This mRNA hybridizes to the heavystrand of Xp2 DNA (11). The inserted AvaI3-PstI3 fragment of the pheS probe was in thesame orientation as in Xp2, and pBl mRNA wasfound to hybridize specifically to the heavystrand of XpS5. For the thrS and pheT probephages, the inserted AvaIl-AvaI2 and BamHI1-SacII3 fragments were in the orientation oppo-site to that in Xp2, and pBl mRNA was found tohybridize specifically to the light strand of XpU6and XpT3 (data not shown).The results of the Southern blotting and the

separated strand hybridization lead us to believethat, although the probes do not show the exact

DNA structures expected, additional DNA isonly of A origin and the phages are true probesfor the pheS, pheT, and thrS genes.Measurement of mRNA levels in plasmid-car-

rying strans. mRNA, pulse-labeled with [3H]uri-dine, was isolated from IBPC2001 carrying pBland other derived plasmids as described above.This mRNA was hybridized to the relevantseparated single strand of Xp2, which carries allthe genes of pBl (13), and the gene-specificprobes XpT3, XpS5, and XpU6. The results aregiven in Table 2. As the sizes of the gene-specific fragments cloned are different, thecounts hybridized were first corrected to countsper kb of DNA. These values were then normal-ized to the values for thrS.The mRNA hybridizing to Xp2 gives the

mRNA specific for the whole thrS to pheTregion. Depending upon growth conditions, thiscan approach 30%o of the total pulse-labeledRNA. This shows what a major effect the pres-

VOL. 152, 1982

on June 14, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

666 PLUMBRIDGE AND SPRINGER

ence of pBl has on the synthesis of transcriptscorresponding to that region of the chromo-some. The molar ratios ofpheS, T to thrS mRNAin pBl-carrying strains appeared to change in a

systematic manner with growth conditions (seebelow), but in all cases, they were of comparableintensities. It is worth noting that the value forpheT was always less than that for pheS, aspredicted by the natural polarity present in apolycistronic operon.The fact that mRNA levels are normalized to

the thrS mRNA level, whose wild-type allele ispresent on all of the plasmids, eliminates any

effect of the change in copy number of theplasmids. The presence of the ilpheSplOOl mu-tation (a cis-acting insertion before the structuralgenes for phenylalanyl-tRNA synthetase, carriedby pBlS caused a major decrease in the level ofpheS, T mRNA, to about 10% of its level in pB1-carrying strains. The ApheS1002 mutation car-ried by pB1T, which produced a somewhatsmaller small subunit of phenylalanyl-tRNAsynthetase, also produced a relative decrease inpheS,T mRNA compared with thrS. Possiblereasons for this effect are discussed. Confirma-tion that structural mutations can affect the levelof mRNAs synthesized is shown by the analysisof pB21-carrying strains. This plasmid is an in-vitro-constructed deletion of the BamHIl toBamHI2 fragment of pBl. This plasmid stillcomplements pheS mutations, but the COOH-terminal part of pheT has been removed. Thedeletion removes the part ofpheT correspondingto the DNA cloned in the pheT probe; thus, nopheT mRNA was detected. However, the valueof pheS mRNA detected was reduced by anamount comparable to that in pB1T, i.e., to aquarter of the level in pBl.The data in Table 2 were obtained under two

different sets of growth conditions: the MOPSmedium (9) supplemented with only the requiredamino acids (conditions A of Table 2) or supple-mented with all of the amino acids (8) (condi-tions B of Table 2). There was a reproducibledifference in the levels of mRNA obtained. Thelevel of mRNA hybridizing to Xp2 was muchlower in the presence of all of the amino acids(conditions B). This could partially be accountedfor by some general effect, such as a decrease inthe mRNA-to-rRNA ratio when the growth rateincreases in the presence of all of the aminoacids. However, a gene-specific differential ef-fect was apparent, namely, that the addition ofall of the nonessential amino acids, includingphenylalanine and threonine, decreased prefer-entially pheS,T relative to thrS mRNA. We arepresently investigating whether this decrease ofthe pheS, T-to-thrS mRNA ratio can be associat-ed with a single or a particular subclass of aminoacids.

DISCUSSION

Different regulation of expression of thrS andpheS,T from pBl. Measurements of aminoacyl-tRNA synthetase activity in strains carrying pBlshowed that the steady-state levels of phenyl-alanyl- and threonyl-tRNA synthetases are en-hanced by factors of 100 and 10, respectively,compared with pBR322-carrying strains (12).The 10-fold difference in the extent of phenylala-nyl- and threonyl-tRNA synthetase overproduc-tion from pBl is not reflected in the mRNAlevels. The results given in Table 2 show that,despite an apparently systematic variation withthe growth medium (see above), the mRNAlevels for thrS and pheS,T (as revealed by a 2-min pulse-labeling) did not vary by factors great-er than two. This result suggests that the thrSand pheS,T promoters are of equal strength andthat the different levels of threonyl- and phenyl-alanyl-tRNA synthetases are the result of post-transcriptional regulation. In agreement withthis interpretation is the observation that plas-mid pBlM1, an in vivo deletion derived frompBl which expresses phenylalanyl-tRNA syn-thetase from the thrS promoter, causes aboutthe same level of phenylalanyl-tRNA synthetaseoverproduction as pBl (12).This would appear to exclude promoter

strengths or specificities as the basis for thedifferential phenylalanyl- and threonyl-tRNAsynthetases synthesis. In an experiment wherepB1-carrying strains were labeled with [3H]uri-dine for 10 min, the pheS-to-thrS mRNA ratioincreased to 1.8 under growth conditions wherea 2-min pulse gave a ratio of 1 (Table 2). Thissuggests a somewhat longer half-life for thepheS,T mRNA. However, this alone does notseem sufficient to explait the factor of 10 differ-ence between phenylalanyl- and threonyl-tRNAsynthetase overproduction.Another possible reason for the difference

between the overproduction of these two en-zymes from pBl could be that threonyl-tRNAsynthetase is less stable than the phenylalanineenzyme. We attempted to compare the stabilityof the two proteins in IBPC2001(pBl) by a pulse-chase experiment as described for phenylalanyl-tRNA synthetase in pBl and pBlT (12). Thisexperiment (data not shown) did not reveal anyclear difference in the stability of the two synthe-tases. Although a small difference in stabilitycould not be excluded, it is certainly not sopronounced as that observed for the wild-typeand the mutated phenylalanyl-tRNA synthetaseof pB1T. Several other possibilities of post-transcriptional regulation exist, e.g., the rate oftranslational initiation or elongation, whichcould produce the different levels of phenylala-nyl- and threonyl-tRNA synthetase observed.

J. BACTERIOL.

on June 14, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

E. COLI PHENYLALANYL-tRNA SYNTHETASE OPERON

These are presently under investigation. Possi-bly the overall 10-fold difference is the result ofseveral small effects.Evidence for autoregulation of the pheS,T oper-

on. Of several spontaneous mutants of pBlisolated, two were studied in more detail, andthe mutations carried by them were classified asregulatory (fQpheSpl 001) and structural(ApheS1002). The plasmid carrying the regula-tory mutation, pB1S, contains a 1.3-kb insertionin front of the structural genes of the pheS,Toperon. The insertion was shown to act in cis,eliminating phenylalanyl-tRNA synthetase over-production. Table 2 shows that mRNA for bothpheS andpheTwas drastically reduced in strainscarrying pBlS compared with thrS. This is thepredicted result for a cis-acting insertion whichis either within the promoter or separating struc-tural genes from their promoter and confirms theclassification of pBlS as carrying a regulatorymutation.Plasmid pBlT carries a small deletion (20 to 30

bp) within the pheS structural gene and pro-duces a small subutnit whose molecular weight isabout 1 kilodalton (kd) less than that of the wild-type subunit and is less stable. As this mutationwas thought to be purely structural, it wasunexpected to find that mRNA levels for bothpheS and pheT are about a third of those foundfor pBl. A possible explanation for this effectwould be a premature termination in the transla-tion ofpheS, inducing polarity in pheT. Howev-er, this explanation does not fit too well with thedata, since both pheS and pheT are reducedproportionately, whereas classical polarityshould induce a greater decrease in the distalpheT mRNA.The most probable cause of the reduced size

of the pBlT small subunit of phenylalanyl-tRNAsynthetase is that the deletion of 20 to 30 bpwithin the AvaI3-PstI3 fragment produces an in-phase fusion, eliminating 7 to 10 amino acids.The molecular weight of the mutated subunit,measured from sodium dodecyl sulfate-poly-acrylamide gels, is about 1 kd less than that ofthe wild-type subunit, which is consistent withthe loss of up to 10 amino acids. There exists,however, the formal possibility that the deletionin pBlT is not in-phase and results in prematuretermination at a normally out-of-phase termina-tion codon. The DNA corresponding to the pheSgene has been sequenced (G. Fayat, C. Sacer-dot, S. Blanquet, personal communication). In-spection of this sequence makes this possibilityvery remote. The pheS gene finishes 216 bp afterthe PstI3 site. Withip this 216-bp sequence,termination codons exist in both of the otherreading frames and would result in the loss of atleast 4 kd in one reading frame add even more inthe third reading frame. Hence, we are sure that

the deletion within pheS ofpBlT produces an in-phase fusion; hence, it is unlikely that there is apolarity effect.We are thus forced to conclude that a defect in

the small subunit of phenylalanyl-tRNA synthe-tase has an effect on the transcription of thepheS, T operon. An analysis of the mRNA frompB21 shows that a defect in the large subunit hasa similar effect. The in vitro deletion ofBamHIl-BamHI2 from pBl to give pB21 removes theCOOH-terminal part of the pheT gene (includingthe DNA corresponding to the pheT probe).The measurement of mRNA levels inIBPC2001(pB21) shows that the pheS mRNAlevel was reduced to a quarter of its value in pBl(Table 2). For this strain, the reduction inmRNA of a promoter-proximal gene caused by adeletion at the end of the distal gene cannot beexplained by polarity.Thus, a mutational alteration of either subunit

causes a decrease in the mRNA levels to about athird of that observed with a wild-type plasmid.Plasmids, which carry only one of the genes ofthe pheS,T operon, do not overproduce thesingle subunit, but the synthesis of the individualsubunits at appreciable levels does take place (asseen by the gel electrophoresis of [35S]methio-nine-pulse-labeled cultures). For pB1T, thesmall mutated subunit is less stable than thewild-type large subunit (12), which should resultin an excess of the large subunit. Thus, twomodels can be proposed for the effect of phenyl-alanyl-tRNA synthetase on its own mRNA lev-el. Either the wild-type tetrameric protein posi-tively regulates its own mRNA level, or theexcess synthesis of one subunit of phenylalanyl-tRNA synthetase negatively regulates themRNA level. At what level this regulation isexerted is unknown; the most likely possibilitiesare either the rate of synthesis or the stability ofthe mRNA. One preliminary experiment showedno appreciable difference in the stability ofpheS,T mRNA from pBl- and pBlT-bearingstrains. The mechanism of action still remains tobe determined, but it is perhaps more likely to beexerted at the level of synthesis of mRNA.These problems merit, and are receiving, furtherinvestigation. We simply state here that evi-dence for some autoregulation of the pheS, Toperon has been observed in strains bearing theoperon on multicopy plasmids. Autoregulationat the transcriptional level has been observed forthe in vitro synthesis of alanyl-tRNA synthetase(14). Our results indicate that a transcriptionalregulation may take place with the pheS, T oper-on. However, our in vivo results do not suggestthe same mechanism. Since the overproductionofphenylalanyl-tRNA synthetase caused by pBlis on the order of 100, negative autoregulation bythe whole enzyme, as claimed for alanyl-tRNA

VOL. 152, 1982 667

on June 14, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

668 PLUMBRIDGE AND SPRINGER

synthetase (14), seems unlikely. Transcription isapparently not the unique control point for thesynthesis of aminoacyl-tRNA synthetases, sincethe differential expression of phenylalanyl- andthreonyl-tRNA synthetases from pBl seems tobe the result of post-transcriptional regulation.

ACKNOWLEDGMENTSWe acknowledge the continued interest and support of

Marianne Grunberg-Manago in this work. We thank GuyFayat, Christine Sacerdot, and Sylvain Blanquet for commu-nicating the pheS sequence data before publication and Mau-rice Hoffnung and Ron Davis for gifts of bacteria and bacterio-phage strains.

This work was supported by grants from the Centre Nation-al de la Recherche Scientifique (Groupe de Recherche 18,A.T.P. "Biologie Mol6culaire du Gene," and "Microbiologie1979") and the D616gation G6n6rale a la Recherche Scientifi-que et Technique (convention 80.E.0872).

LITERATURE CITED1. Blattner, F. R., B. G. Williams, A. E. Bkechl, K. Dennison-

Thompson, H. E. Faber, L. A. Furlong, D. J. Grunwald,D. 0. Klefer, D. D. Moore, J. W. Schuimm, E. L. Sheldon,and 0. Smithies. 1977. Charon phages: safer derivatives ofbacteriophage 1 for DNA cloning. Science 196:161-169.

2. Blumenthal, R. M., P. G. Lemaux, F. C. Neidhardt, and P.P. Dennis. 1977. The effect of reUA gene on the synthesis ofaminoacyl-tRNA synthetases and other transcription andtranslation proteins in Escherichia coli B. Mol. Gen.Genet. 149:291-2%.

3. Comer, M. M., and A. B&ck. 1976. Genes for the a and osubunits of the phenylalanyl transfer ribonucleicacid synthetase of Escherichia coli. J. Bacteriol.127:923-933.

4. Davis, R. W., D. Botsteln, and J. R. Roth. 1980. Advancedbacterial genetics. Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.

5. Hagen, F. S., and E. T. Young. 1978. Effect of RNase IIIon efficiency of translation of bacteriophage T7 lysozymemRNA. J. Virol. 26:793-804.

6. Miller, J. H. 1972. Experiments in molecular genetics.Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.

7. Nam, G., and F. C. Neidhardt. 1967. Regulation of forma-tion of aminoacyl-transfer ribonucleic acid synthetases inE. coli. Biochim. Biophys. Acta 134:347-359.

8. Nekdhardt, F. C., P. L. Bloch, S. Pedersen, and S. Reeh.1977. Chemical measurements of steady-state levels of 10aminoacyl-transfer ribonucleic acid synthetases in Esche-richia coli. J. Bacteriol. 129:378-387.

9. Nekdhardt, F. C., P. L. Bloch, and D. F. Smith. 1974.

Culture medium for enterobacteria. J. Bacteriol. 119:736-747.

10. Nddhardt, F. C., J. Parker, and W. G. McKeever. 1975.Function and regulation of aminoacyl-tRNA synthetasesin prokaryotic and eukaryotic cells. Annu. Rev. Micro-biol. 29:215-250.

11. Phumbrklge, J. A., and M. Springer. 1980. Genes for thetwo subunits of phenylalanyl-tRNA synthetase of E. coliare transcribed from the same promoter. J. Mol. Biol.144:595-600.

12. PJbrldge, J. A., and M. SpInger. 1982. Escherichiacoli phenylalanyl-tRNA synthetase operon: characteriza-tion of mutations isolated on multicopy plasmids.J. Bacteriol. 152:650-660.

13. Plmbrldge, J. A., M. Springer, M. Graffe, R. Goursot,and M. Grunberg-Manago. 1980. Physical localisation andcloning of the structural gene for E. coli initiation factorIF3 from a group of genes concerned with translation.Gene 11:33-42.

14. Putney, S. D., and P. Schrmnel. 1981. An aminoacyl-tRNA synthetase binds to a specific DNA sequence andregulates its gene transcription. Nature (London)291:632-635.

15. Smith, D. I., F. R. Blattner, and J. Davis. 1976. Theisolation and partial characterization of a new restrictionendonuclease from Providencia stuartii. Nucleic AcidsRes. 3:343-353.

16. Springer, M., M. Graffe, and M. Grunberg-Manao. 1979.Genetic organization of the E. coli chromosome aroundthe structural gene for initiation factor IF3 (iqfC). Mol.Gen. Genet. 169:337-343.

17. SprInger, M., M. Graffe, and H. Hennecke. 1977. Special-ized transducing phage for the initiation factor IF3 gene inE. coli. Proc. Natl. Acad. Sci. U.S.A. 74:3970-3874.

18. Springer, M., J. A. Plumbridge, M. Trudel, M. Graffe,and M. Grunberg-Manago. 1982. Transcription unitsaround the gene for E. coli translation initiation factor IF3(inJ). Mol. Gen. Genet. 186:247-252.

19. Sutllfe, J. F. 1979. Complete nucleotide sequence of theEscherichia coli plasmid pBR322. Cold Spring HarborSymp. Quant. Biol. 43:77-90.

20. Szybalsk, E. H., and W. Szybalski. 1979. A comprehen-sive molecular map of bacteriophage lambda. Gene 7:217-270.

21. Yamamoto, K. R., B. M. Alberta, R. Bea_nger, L. Law-borne, and G. Treiber. 1970. Rapid bacteriophage sedi-mentation in the presence of polyethylene glycol and itsapplication to large scale virus purification. Virology40:734-744.

22. Yanofsky, C. 1981. Attenuation in the control of theexpression of bacterial operons. Nature (London)289:751-758.

23. Z -nrerran, S. B., and G. Sandeen. 1966. The ribonucle-ase activity of crystallized pancreatic deoxyribonuclease.Anal. Biochem. 14:269-277.

J. BACTERIOL.

on June 14, 2018 by guesthttp://jb.asm

.org/D

ownloaded from