Structural arrangement of tRNA binding sites on Escherichia coli ribosomes, as revealed from data on affinity labelling with photoactivatable tRNA derivatives

BBAEXP 91954

Biochimica et Biophysica Actag 1008 (1989)146-!56 Elsevier

Structural arrangement of tRNA binding sites on Escherichia coli ribosomes, as revealed from data on affinity labelling

with photoactivatable tRNA derivatives

Dmitrii M. Graifer, Galina T. Pab)aa, Natalya B. Matasova, Sergei N. Vladimirov, Galina G. Karpova and Valentin V. Vlassov

Institute of Bioorganic Chemistry, Siberian Division of the Acaderny of Sciences of the U.S.S.R., Novosibirsk ( U. S, S. R.)

(Received 19 September 1988) (Revised manuscript received 26 January 1989)

Key words: tRNA binding site; Photoaffinity labeling; Ribosomal protein: (E. coil)

A systematic study of protein environment of IRNA in ribosomes in model complexes representing different translation steps was carried out ttsing the affinity lahelling of the ribosomes with tRNA derivatives bearing aryl azide groups scattered statistically over tRNA guanine residues. Analysis of the proteins crossfinked to tP,~4A derivatives showed that the location of the derivatives in the aminoacyl (A)site led to the labelling of the proteins $5 aim $ 7 in all complexes studied, whereas the labelling of the proteins $2, $8. $9, S l l , S14, SI6, S17, SIR, $19, $21 as well as L9, LII, LI4, L15, L21, L23, L24, L29 depended on the state of tRNA in A Site. Similarly, the location of tRNA derivatives in the peptidyl (P)site resulted in the labelling 0 f the proteins L27, $11, SI3 and $19 in all Mates, whereas the labulling of the proteins $5, $7' $9, SI2, S14, $20, S21as well as L2, LI3, LI4, LI7, L24, L27, L31, L32,L33 depended on ~ ~Tpe of complex. The derivatives of tRNA Met were found to crosslink to Sis 83, $5, ST, $9, S14 and L I , L2, LT/LI2, Based on the data obtained, a generall principle of the dynamic functioning of r i ~ has been proposed: (i) the formation of each type o f ribosomal complex is accompanied by changes in mutual ~ n t o f prote ins- 'conformational adjustment' of the ribosome - and (ii) a ribosome can dynamically change its internal strueture at step o~' ~nitiation and elongation; on the 70 S ribosome there are no rigidly fixed structures forming tRNA-binding sites (primarily A and P sites).

Introduction

B y now, all steps of initiation and elongation of the polypeptide chain on ribosomes have been studied in detail. However, the molecular mechanisms of associated processes, e.g., translocation, remain as yet rather vague. To understand these mechanisms, it is necessary to know whether (and to what extent) the interaction of the translation participants, par';~ularly of tRNA, with ribosomes can change at eac Lep of translation. To investigate the tRNA,ribosome interaction; a variety of simplified model Systems (e.g, non-enzymatic F, inding o f tRNA and mRNA: the N-acetylphenylalanine re- Sidue as a model of the peptidyl residuel etc.) are often used~ It is not clear, however, whether the location of : tRNA on the ribosome is identical for the various types

Correspondence: D.M. Graifer, Institute of Bioorganic Cn.::~istry. Siberian Division of the Academy of Sciences of the U.S.SR., La),:r~ntiev Prospekt 8, Novosibirsk 630090, U.S~S,R.

r o c;~ ~e, ~ ~lqg~ Eisevier Science Publishers BAL (Biomedical

of complex representing the same ribosomal state. A possible difference in tRNA location may be the reason why the data reported by different authors are some- times conflicting.

The mos'~ informative approaches to the study of functional topography of tRNA on ribosomes are based on covalent attachment of tRNA to ribosomes with subsequent analysis of ribosomal components crosslinked to tRNA. Among these approaches are (i) d i r~ t ultraviolet-induced (Tt = 260 nm) crosslinking of tRNA to the proteins [1-4], as well as crosslinking of rare minor bases of some tRNAs to 16 S r R N A u p o n softer ultraviolet irradiation [5], and (ii) affinity labC~mg o f ribosomes with tRNA derivatives bearing:an active (usually photoactivatable) group at a given positien [6--8]. The two methods are complementary, each hav: ing its limitations.

T h e former approach was used t o examine the protein environment of tRNA molecules in complexes imitating different steps of the elongation cycle [4] ~md the 3f~ S ~.:omplex initiation [2]; upon soft uRraviolet-irradia;

Division)

tion it was possible to detect the crosslinking of minor 5"-anticodon bases of some tRNAs to the 16 S rRNA Cl400 residue at the ribosomal P site [9]. The application of the affinity labelling method has been hindered so far by a limited set of tRNA ~ites in which reactive groups can be introduced. For example, Phe-tRNA phi, having a n aryl azide group on the 4-thiouridine residue at the A site, was found :to crosslink to the S19 protein [10]. T h e N-ACPhe-tRNA r~ derivative with thiolated C3, and C6o residues, was attached to the SI0 protein at the P s i te upon irradiation [8]. Finally, the photocrosslinking of theE. coil tRNA TM via an aryi azide group

on 5,-anticodon base (5-carboxymethoxyuridine) to the C14o0 residue of 16 S rRNA at the A si:e was also reported [nl .

:The present paper reports the results of a systematic study of topography of tRNA molecules on ribosomes in the most common model complexes imitating various steps of initiation and elongation (with different extent of approximation to the real translating system). Two types of tRNA derivative with t4C-labelled photoactivatable groups, scattered over the tRNA molecule (primarily over atoms N-7 of guanosine residues), namely, azido-tRNAs, were used. The preparation and application o f such tRNA derivatives have been described :[12-14], The :preparation procedure falls into two steps: alkylation o f tRNA with 4-(N.,?-chloroetbyl-N-methylamino)[~4Clben~ylamine (CIRCH2NH2), and subsequent treatment with 2,4-dinitro-5-fluorophenylazide. Thus, a selective attachment of:aryl azide groups to aliphatic amine groups of RCHzNH 2 residues takes place. Using these derivati~,es, it appeared possible to' detect differences in protein environment of tRNA both at different tRNA-binding sites of ribosomes and in different states of the same tRNA-bi~iding site (P or A).

iMaterials and Methods

tRNA-~ et (1200 pmol/A260 unit) (Boehringer-M~n- nheim); poly(U)~ ATP and GTP (Reanal, Hungary); [laC]phenylalanine (318 mCi/mmol) (UVVVR, Czecho- slovakia); elongation factors Tu-Ts (3000 pmol/mg) and total factor Tu. Ts + G (13 mg/ml) were obtained as in Ref.: 15; :initiation factors IF-I, IF,2 and IF-3 and the fragment ot MS2 phage RNA with a length of 56 micleotides bearing the initiation AUG codon were obtained as described i n Ref. 16 and kindly donated by ~'oL E,Ya.: Gren: i Ribosomes were isolated from E . coli MRE-600 according toReL 17. For the experiments with EF-Tu- dep::~adent binding of Phe,tRNAP~ and IF-2-dependent binding of fMet, tRNAr~! and their derivatives, ribosomes were obtained as in Ref. 18 and kindly donated by Dr, V.I, Makhno. The activity of ribosomes in poly(U)-dependent nonenzymatic binding of Phe-

147

tRNA Phe was 50 and up to 90% in the former and latter case. respectively.

Enriched preparations of Phe-~RNA Phe (1400 pmol/ A,_60 unit), AcPhe-tRNA Ph¢ (1400 pmoi/A2~ ~ u,~it),

Phe AcPhe-tRNAo~.~e a (I250 pmol/A,~) unit) and deacy!- ated tRNAPo~ were isolated as in Refs. 18, 19 and kindly donated by Drs. Yu.P. Semenkov and V.B. Odintsov.

4-(N-2-Chloroethyl-N-methylamino)[~4C]benzylam- ine (CIRCH2NH2) with a specific activity of 25 mCi/mmol was obtained according to Refs. 20, 21. To prepare photoactivable derivatives, tRNA P~ or tRNA~ et were first alk>!ated with CIRCH:NH 2 (generally to the extent of about 2 mol of the reagent residues per tool of tRNA) and then treated with 2,4-dinitro-5-fluorophenylazide, as described in Refs. 12-14. If necessary, the extent of tRNA modification gas altered by changing the incubation time with CIRCH,NH 2. When the alkylation was performed under conditions of tRNA tertiary structure lability (0.2 M NaCI/0.01 M EDTA/0.03 M NaAc (pH 6.0), 37°C), azido-tRNA-I was obtained; under conditions of tRNA tertia~ structure stability (0 .2M NaCi/0.01 M MgCI2/0.03 M NaAc (pH 6.0), 20°C) azido-tRNA-ll was obtained.

Aminoacylation of tRNA Ph~ and azido-tRNA Ph¢ was carried out in standard conditions using phenylalanyl- tRNA synthetase of 85~ purity grade (according to the polyacrylamide gel electrophoresi~ data) kindly donated by Dr. V.N. Ankilova; fMet-tRNA~ ~' and fMet-azido- tRNA~ e~ were obtained by aminoacylation of the corresponding preparations with the aid of S-100 E. coil supe-natant fractionated with streptomycin sulfate and 40-50% ammonium sulfate; N-10-formyitetrahydrofolic acid was applied as formyl group donor [22].

Aminoacylated and formylated tRNAs were isolated from reaction mixtures using ion-exchange chromatog- raphy on DEAE-cellulose followed by precipitation with ethanol. The extent of aminoacylation and formylation of tRNAs was determined as in Ref. 23.

Binding of tRNA to ribosomes Non-enzymatic binding of tRNA Ph~ derivatives to

ribosomes ((2-3). 10 -6 M) was performed in the presence of poly(U) (1 mg/ml) at 0°C in the A, buffer containing 0.1 M NH4CI and 0.05 M Tris-HCl (pH 7.5) (x = concentration of MgCI 2, mM). Incubations were carried out in a final volume 1-5 ml for l h. EF-Tu-dependent :binding of Phe-tRNAeh¢ derivatives to ribo- ~,omes was performed in A~0 buffer in two steps. First, the ternary complex (1), namely, Phe-tRNA Ph¢ • Tu- GTP

was obtained by a 15 min incubation of the mixture of tiaese components at 0 o C, the respective concentrations of the above compounds being 3.10 '-~ :M, 2.10 -~ M (EF-Tu. 'Is) and 2.10 -4 M.: In some experiments GTP was replaced with its non-hydrolizabte analog, GMP-fl, y-methylenediphosphonate (GMPPCP) tServa,

F.R.G0 in 1.2- 10 ":3 M coiice~atration and EF-Tu - Ts - with free EF-Tu (1 .10 -5 M) obtained as in Ref. 24. Final v.~umes were 1-4 ml. Second, the complex (1) was incu ~ate0 (~i o C, 15 min) with a complex ribosome- po ly ( U) Act~e-tRNA Phe (or poly(Phe)-tRNA Phe, see below) taken in approximately equimolar amounts with respect to Phe-tRNA Phi" in the complex (I). In some experiments either deacylated :tRNA ere or Ac-Phe- tRNAP~[~r~ d (with a broken C2' -C3 ' bond in the ribose of 3'-end adenosine this analog of peptidyl-tRNA cannot participate i n the transpeptidation reaction on ribosomes [191) was used: Binding o f a non-modified tRNA: to ribosomes was examined b y nitrocellulose filtration technique, and that of [14C]azido-tRNA by gel filtration on Sephadex G-200 or centrifugation of complexes: through 10% sucrose (see below).

In some experiments, antibiotics like tetracycline (Serva) a n d edeine (Calbiochem, U.S.A.) with a final concentration 3 .10 -s M were used; in these cases ribosomes were preinc~,~ated with antibiotics for 30 min at O°C,

To obtain the post-translocationl complex, ribosome. poly(U) (Phe),-azido-tRNA phi, a mixture of 70 S ribosome:~ (2.3 - 10 -6 M), Phe-azido-tRNA r'h~ (7.5.10 -6 M), poly(U) (1.3 mg/ml) and the total elongation factor (6 mg/mi) was incubated for I h at 37°C in the A10 buffer containing 0.3 mM GTP (conditions :for the completion of poly(phenylalanine) synthesis. Then the complex was isOlated by centrifugation through 105g sucrose in the same buffer but free of GTP (105 000 × g, 40C, 4 h). Upon incubation of the isolated complex with Phe-tRNA : Tu . GTP, the pretranslocation complex (Phe),+l-tRNA~h ~ (A site) .r ibosome, poly(U). az ido - tRNA~ (P site):was obtained. Incubation of the pos[-translocation complex with I mM puromycin (Serva) for 40 min at 0 ° C yielded the complex ribosome • poly(U)- azido-tRNA~"~ (P site). Both the complexes were isolated as described above. Evidence for the correct location of the tRNA derivatives in pre- and posttranstocation complexes are given in Table IV and discussed in the Results section. The 70 S initiation complex, fMet-azido-tRNAM~t-70 S ribosome-MS2 RNA, was obtained by incubation of ribosomes (0.65. 10 -6 M) with the MS2 RNA fragment (28 A26o/ml ), fMet-azido-tRNA~ ~ (10 `7 M), IF-1 (0,05 mg/ml), IF-2

(0 .25 m g / m l ) a n d 1F-3 (0.(K~8 mg/ml) factors in the A 5 buffer Containing 0.2 mM GTP for 10 min at 37°C.

i

Irradiation of complexes and analysis of products of az!do-~RNA attachment to ribosomes

Complexes were irradiated with ultraviolet light of a high-pressure mercury lamp (1 k W ) i n a flow glass cuyette-capillary with about 2 mm inner diameter which was Cooled with water to + 120 C (X > 310 nm, glass filter).: The reaction mixture flow rate was 2-4 ml/h. The distribution Of the labeiled azido-tRNA crosslinked

i i

:

. . . . . :

to ribosomes over subunits was analysed by centrifugation of the irradiated complexes in sucrose concentration gradient (10-30%.) under dissociating conditions (buffer A 0.5). Optical absorbance ar, d radioactivity were determined in all fractions,

To analyse the distribution of azido-tRNA cross, linked to subunits between rRNA and proteins~ t h e isolated modified subunits were centrifuged in sucrose l concentration gradient ( 5 - 2 0 % ) i n the presence of sodium dodecylsulfate and EDTA [6],

Ribosomal proteins with crosslinked azido-tRNA were isolated together with non-modified proteins from the irradiated complexes by extractio=J in 2 M LiCI with 4 M urea (100 h, 4 o C) [25]. The sapernatant containing ribosomal prote2aS was dialyzed against :the buffer c o n - taining 3 M urea, 0 . 0 I M EDTA and 0.01 M Tris-HCl (pH 7.5). To remove the covalently attached tRNAS; the modified proteins were treated with large excesse s o f RNases A and T~ and alkaline phosphatase [26]. Then the sample was dialyzed against buffer (7 M ureai 0.02 M Tris-H3BO 3 (pH 8.6)) and concentrated against 30% poly(ethylene glycol) in the same buffer t o achieve a 40-100 #1 vohimel

The proteins were analysed by two-dimensional elec- trophoresis in polyacrylamide gel as described in Ref. 2Z The spots corresponding to the proteins and gel pieces around the spots were cut out, and the proteins were extracted froni the gel by 0.5% sodium dodecy! 7 sulfate for 24 h at 40 o C. The radioact;vity was counted ir~ a dioxane scintillator. The amount of 70 S ribosomes • .::ken in one experiment for identification of the mod ' ified proteins was 100-200 A260 units.,

Results

Functional activi O' of azido-tRNA It is evident that the greater the extent of azido-tRNA

modification (n), the less is its functional activity. This is clearly seen from Table I. The fall in acceptor activity has also been observed before for various modifications of tRNA p~ [28]. Azido-tRNA-ll has a higher acceptor activity than azido-tRNA-I with the same extents of modification (Table I). This is not surprising, since in the case of type II preparations there are no aryl azide groups on the nucleotide residues cSvential for the f o r - mation of the IRNA ter t iary structure [12,29], i .e . , azido-tRNA-lI is less different from the native t R N A than azido-tRNA-l.

For determining of the [14C]azido-tRNAPhe ( n = 2) activity in non-era.ymatic poly(U)-dependent binding to ribosomal P site the tRNA derivatives were titratecl with the ribosomes in buffer A~0 (at110 imM Mg: , template-free tRNA binding is almost negligible). The maximum binding of both tRNAonon derivatives has been observed even at 1.5-fold excess o f an active fraction of ribosomes with respect to a~ido-tRNA a n d

T A B L E l

Maximum extents ,~f ami~ioac),latic;~ (co ~, for ,,arinu~ azi:do-tRNAs

Extent of aminoacyla t ion for the nat ive t R N A Pne is t aken as 100qL

ntzido-tRNA Mol reactive groups per a (%) tool t R N A (n )

a z i d ~ - t R N A r ~ - I 1 70

azido. tRNAPh¢-l l

a z i d o - t R N A ~ L !

azido-t RNA~et - l l

2 50 3 35 4 25

2 75

2 55 4 30

2 95 4 90

achieved up 907o f , r azido-tRNA-ll and 75% for I. Thus, in this system the functional activity of azido- tR.'~iA-lI is also higher than that of I; the relative activity of azido-tRNA ~e in binding to ribosomes being !ao, gher than m armnoacylation system. In other words, ribosomes are less sensitive to the presence of a strange chemical group in the tRNA.

W e have shown previously that a non-enzymatic binding of azido-tRNA eh¢ at the ribosomal P site (both poly(U)-dependent and template-free) observed at a high, up to20 mM, Mg 2+ concentration [14] is specific, i.e., occurs at the same site as the native tRNA ph¢ binding [13,14]. This conclusion follows from the

• i

149

experiments indi,:ating that ~z~do-tRNA Ph" inhibits the [~C]Phe-tRNA Ph¢ binding in the t~resence of tetracycline, which blocks the A site. On the other hand, the native tRNA Ph~ inhibited the binding of [14C]azido- tRNA Ph¢ to the ribosomes [13.14].

Factor-depe~dcnt binding of Phe-azido.tRNA ph~ to ribosomes at the A site or fMet-azido-tRNA~ *t in the 70 S initiation complex is also specific, as follows from the data shown in Table II. For example, the Phe- azido-tRNAPh%l binding is largely dependent on the Tu factor and is almost completely inhibited by 3 . 1 0 - -~ M tetracycline; a,:cording to the criteria adopted, these obsc:'vations pJ~'ove the A site binding. In a similar way, the fMet-azido-tRNA~ et binding in the 70 S initiation complex in buffer A~ requires the presence of IF-2 factor and is inhibited by the native fMet-tRNA~ ~'. The data for various azido-tRNAs with n = 2 are listed in Table II. At n = 3-4, the efficiency of factor-dependent binding of the corresponding derivatives to ribosomes decreases by 25-50%, depending on n and the type of complex. Non-enzymatic binding of azido-tRNA eh* to ribosomes is h.'ss sensitive and changes only slightly with an increase in n up to 4 (data not shown).

The data on the EF-Tu-dependent binding of Phe- azido-tRNAPh%l~ to ribosomes in model complexes imitating the states from recognition of aminoacyl-tRNA up to the formation of the simplest pretranslocation complex are corr~piled in Table II!. In this case. as in a simple model system (Table II), the binding of the tRNA r'h~ derivafi,:te is inhibited by tetracycline and depends on EF-Tu and GTP or GMPPCP.

T A B L E [1

14 Pht Binding and covalent attachment o f Phe-[ C]azido-tRNA (n = 2) at the A site (complex of o,pe 1 ~ and fMet-[ / 4C]azido-tRNA~et (n = 2) in 70 S initiation complex (types 2 and 2a)

Complexes of type 1 were ob ta ined in three steps: (i) formation of a ternary complex Phe-az ido- tgNA e~- E F - T u - G T P (in one case EF-Tu was omit ted) ; (ii) b ind ing of the deacylated t R N A ~ to the P site of po ly(U)-programmed r i l~somes, an:l (iii) incubat ion of the lattel complex with the ternary one as described in Mater ia ls and Methods.

Type Azido IF-2 EF-Tu lnhibi- Binding. Cro~i~tinking of t lkNA tar real t R N A mol t R N A per complex per r to i real subuni ts

r ibosomes 30 S 50 S

1 Phe-azido- ,- + - 0.400 0°055 0.044 tRNArOae-I - - - 0.050

-- + tetra cycline 0.020 0,002 0,002

2 fMet-azido- + - - 0.110 0.025 0.0! 8 t RNA'rMet-I - - - 0.008

+ -- 3 . i 0 -6 M fMeI- tRNA Met 0.027 0.006 0AX)4

2a fMet-azido- + - - 0.150 " 0.048 0.008 tRNA~"t - I 1 - - - 0.01,t

+ -- 3 .10 -6 M

fMet-t R N A ~ ct 0.(}30 0,010 0,002

• This value corresponds to the a lmos : comple te b ind ing of fMet-azido- tRNA, since for the p repara t ion of the 70 S ini t ial on c-mple:~ r bosomcs were in a 6.5-fold excess with respect to fMet -az ido- tRNA~ ~t (,see Materials and ~-")" ~ t~

: / : ~ i ̧:~ ~ i . . . . • !

: r50

TABLE I11

Binding and ctmalent attuchment of [14C]azido.tRNA t,ae-I (n = 3) at the A site h7 different states

The complexes were obtained in three steps: (i) formation of ternaD' complexes Pbe-[14C]azido.tRNAt'he-EF-Tu.GTP(GMPPCP for complex (3)), in ,ome cases EF-Tu and /o r GTP (GMPPCP) were omitted; (ii) binding of AcPhe-tRNA Phc or AcPhe-tRNA~C,~ to the P site of poly(U)-programmed 70 S ribosomes • and (iii) incubation of the latter complex with the ternaD ~ one as described in Materials and Methods.

Modelled state EF-Tu GTP GMPPCP Tetra- Binding, mol CrosslinKing mol cycline azido-tRNA azido-t2.NA

per mol per mol subunits ribosomes

30 S 50 S

(3) Recognition of cod0n-specific aa-tRNA before GTP hydrolysis"

(4) After GTP hydrolysis but before transpeptidation ~'

~5) After transpep- tidafion (pretrans-

:location complex) a

+ - + - 0.240 0.045 0.026 + . . . . 0.070 + - 4 + 0.020 0,004 0.002 . . . . . 0.10

+ + - - 0.210 0.043 0.038 + + - + 0.030 0.005 0.003

+ + - - 0.220 0.044 0.026 + + - + 0.030 0.005 0.002

* T h e binding efficiency for peptidyl-tRNA analogs was 0.9-1.0 too! per tool of ribosomes according to data of parallel experiments with Ac114C]Phe-tRNA r'he and Ac[ 14 ClPhe.tRNAr~ ~ d" AcPhe-tRNA Ph¢ preb~mnd at the P site.

P h e t~ AcPhe.tRNA,,~.r~ J prebound at the P site,

Finally, azido-tRNA Ph~ also works in a more complex system, which is closer to the conditions of the pro!ein synthesis in the cell, ire., EF-Tu- and EF-G-dependent Synthesis of poly(phenylalanine) on the poly(U)

!

template(Table IV)~ After termination of the poly(phenylalanine) synthe-

Sis (a plateau on the curve of [14C]Phe incorporation into an ac'_'d-insoluble fraction in control experiments) (Phe),-azido-tRNA Ph~ is located in the P site, the A site remaining vacant. This conclusion follows from the data on the almost quantitative removal of [14CIpolyphenyl- alanine from the ribosome after the treatment with puromycin (Taole IV, lines 1 and 2). Addition of EF-Tu, Phe-tRNA Ph¢ and GTP to the post-translocation complex; isolated from the reaction mixture, suppresse~ the reactivity of a peptidyl moiety towards puromycin (Ta- ble IV, lines 5 and 6). The above data indicate th, • filling of the A site and formation of the peptide bond, leading eventually to the pretransloca:tion complex of the subsequent elongation cycle. The complex formed has (Phe)~.t-tRNAeh~ a t the A site and deacylated azido-tRNA~h~ at the P site.

Thus. azido-tRNA is suitable for Studying th e topography of thetRNA-binding sitc3 of ribosomes in almost

i i

any model comPlex.

Cot,alent a~tachment of azido-tRNA to ribosomes i The data on; distribution of the crosslinked azido- tRNA betwee n the ribosomal subunits in various model complexes are listed in Tables II, III and V. It can be

seen that, in all cases, botil the subunits undergo labelling which is preferential for 30 S. Such a predominant modification of the 30 S subunit is also typical of azido-tRNAPh%assisted affinity labelling of the ad, ditional binding site of deacylated tRNA on the ribosomes - the 'E site' (the model azido-tRNA r~c. ribosome complex hao beer_ obtained without a template in the presence of edeine blocking the P site [30]) [31]. The only exception is the crosslinking of azido-tRNA exclusively to the 30 S subunit in the double complex with the 70 S ribosome [14]. These results are consistent with the universally adopted idea that tRNA-binding sites of ribosomes are located on contact surfaces of subunits and the A and P sites are forrned mainly by the 30 S subunit [18].

As follows lrom the data in Tables 11, IH and V, the extent of crosslinking of aztdo-tRNA to ribosomes depends on both the type of tRNA derivatives and the complex, and amounts to 20-50%.

Analysis of the distribution of azido-tRNA crosslinked between rRNA and the proteins has not revealed any significant modification of rRNA in any instance (15 S RNA no t tested)~ It Seems very unlikely that, in either of the states studied, the elongated and flexible reacitve groups of azido-tRNA with a maximum length of the spacer moiety up to 13 ~, make contact with any site of 16 S or 23 S rRNA. The inability to modify RNA seems to be due to the insufficient activity of photoactivatable groups. Aryl azid~ derivatives Of tRNA with the other structure of aryl azide groups can Crosslink, f o r exam.

i i

t51

TABLE IV

Binding of azido-tRNAeht-I derivatives (n = 2) to ribosomes upon the termination o/ poly(U)-dependent svmhesis of po(),¢Phe) in the presence of elongatioin factors and GTP

No tRNA P~ derivative Added Binding being bound at P site Phe-tRNA Ph" puro- (tool azido-tRNA upon termination of EF-Tu mycin per tool ribosomes) poly(O) translation GTP before/after irradiation

Amount o f Phe residues bound per ribosome before/after irradiation

l [ 14 C](Phe) .-azido-t RNA - - 2 - +

3 (Phe),,-['4 C]azido-tRNA - 4 - +

5 [ ,4 C](Phe),,-azido-t RNA + 6 + +

7 (Phe),,-[ 14 C]aaido-tRNA +

0.32/0.29 0.29/0.26

0.30/0.27

3.30/3.00 ~ 0.17/0.14

3.0O/2.80 2.90/2.70

a A real average length of poly(Phe) chains equals approx. 10 Phe residues, since the amount of (Phe).-azido-tRNA bound per I mol of ribosomes averages 0.3 mol.

TABLE V

Covalent attachment of [14C]azido-tRNAehe (n = 2) to the 1' site 6f ribosomes in d~fferent states in the presence of poly(U)

Complex a tRNA phe tRNA at Binding of Attachment of azido- derivative the A site azido-tRNA tRNA (mol per mol at the (mol per tool ribosomal subunits)

P site ribosomes) 30 S 50 S

6 azido-tRNA-I - 0,220 0.035 0.020 7 aaido-tRNA-ll - 0.240 0.029 0,014 8 azido-tRNA-ll Fhe-tRNA e"c 0.210 0.022 0,012 9 (Pbe) n-azido- tRNA-I - 0.320 0.120 0,030

10 azido-tRNA-! - 0.290 0.090 0.020 11 azido-tRNA-I (Phe). + I-tRNA Phc 0,300 0.120 0.050

" in complexes (6)-(8) azido-tRNA was bound non-cnzymatically and 3-fold excess of ribosomes over IRNA was used; post-translocation complex (9) was obtained as described in Materials and Methods; complex (10) was obtained by treatment of (9) with puromycin; complexes (8) and (11) were obtained from (7) and (9), respectively, by their incubation with the ternary complex Phe-tRNA Ph¢. EF-Tu .GTP,

ple, to 16 S rRNA in different complexes with 70 S ribosomes [11].

The specificity of crosslinking of azido-tRNA to ribosomes (i.e., attachment in the desired complex to the desired tRN,~-binding site) ha, b~,.A, proven by a number of experiments. In a non-enzymatic system, the crosslinking of azido-tRNA ~h~ to ribosomes is almost negligible if the corresponding site has been preblocked

Phe by a non-modified tI~NA [13,14]. Similarly, a non- modified fMet, tRNA~ e' protects the ribosomes from niodificatiot: with azido-tRNAf Me' in the 70 S itfitiation complex (Tabie If). The covalent attachment of Phe- azido-lRNA eh¢ to tbe ribosomal A site is EF, Tu-dependent and is blocked in all cases by 3.10-5 M tetracycline (Tables II and 111). All these data prove that the extent of nonspecific (beyond the complex) modifica- t ionof ribosomes with azido-tRNA under the employed conditions is negligibly small Finally, irradiation has l i t t le effect on the location of azido-tRNA on the ribo-

z i

Fig. 1. Typical electrophoregram of the proteins isolated from irradiated ribosomes in ~.he complex with azido-tRNA.

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200

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2 lt. : 3C0 ~00 200

PROTEIN NU~8[~ PRo'r(IN NOl~t~

Fig. 2 Distribution of ~4C label over ribosomal proteins labelled with azid0-tRNAO~-I at different steps of EF-Tu-dependent binding at the A Site of 70 S ribosomes. (1) complex (3): Phe-[t4C]ezido-tRNA e~. EF-Tu.GMPPCP.70 S-poly(U)-AcPhe-tRNA r'~ ~P ::,te); (11) complex (4): Phe-[]4C]azido-tRNA ehe (A site).70 E'poly(U)-AcPhc-

Phe tRNAo~.r~ ( P site); (111) complex (5): AcPbe-Phe-[ ]'~CIe~Ado-tRNA '~* (A s t ,e )70 S . p o l y ( U ) . t R N A ~ (P site). See Table ill.

,~00 T :,llO % H

L 2{h?

5 ~ 13 2 ~. 5 I0 ~5 L) ~ A F~J;E*N NJ¢~uB£~

Fig, 3, Distribution Of t'*C label over ribosomal proteins labelled with [14C]azid(~-tRNAo~ bound at the P site. (1) complex (6): azido- tRNAo~-I.70 S,p01y(U); (ll) complex (7): azido-tRNA~-II-70 S. p~ly(U); (11!) complex (8): Phe, tRNA I~ (A site),70 S.poly(U).

azido-tRNA~t-lI (P site). See Table V.

Fig. 4. Distribution of 14C label over ribosomlil proteins latmlled with i [14 C]azido.tRNAP~_l bound at the P site upon termination of poly(U) translation. (1) complex (9): (Phe),-azido-tRNt-. I~- 70 S. pu,y,.,-,j,-" " '~- '~!,pl~ complex (10): azido-tRNAo~-70 S. poly(U) obtained by treatment of complex (9) with puromycin; (Ill) complex (11): (Phe),,+ rtRNA Ph¢. 70 :'~-poly(U)-azido-tRNAo~no8 obtained by incubation of complex ( 9 )

with the ternary complex Phe-tRNA t~e. EF-Tu. GTP. Table V.- :

some. This can be deduced from the data g iven in Tab l e IV. The extent of b ind ing o f a z i d o - t R N A ~¢ and i t s reactivity towards p u r o m y c i n alter ins ign i f i cant ly in post- and pretrans locat ion states u p o n ircadiat ion o f the corresponding complexes .

Ribosomal prozeins crosslinked to azido-tRNA in various complexes

A typical e l ec trcphoregram of 70 S r ibosomal proteins from the irradiatsd cumv,~,,,;~- -' . . . . . . is d i sp layed in Fig.

CPM 305 ( ~ ) SOS

250 A ;50

5UD ~ 500 j

5 10 ~5 20 5 I~ 15 ZO 25 ? 30:

~ROTEiN NUHe['e :

Fig. 5. Distribution of I4C label over ribosomal proteins labelled With 14 Met fMet-[ Clazido-tgNA t -1 (A) and fMet-[:14Clazide~-tRNAM~-li (B)

in the 70 S initiation complex. See Table II (~rrest~nding complexes 2.2a.

~53

1. It is seen ~hat almost all the ribosomal proteins are separated satisfactorily. We have shown earlier that hydrolysis of azido-tRNA in large excess of RNases A and T~ and alkaline phozphatase produces di- and /o r trinucleotide fragments, which have 0 or - 1 charge (provided by the charge +1 on atoms N-7 of the modified guanine residues), hardly affec s the electro- phoretic mobility of the ribosomal proteins [26]. There- fore; the modified proteins can be identified quite relia- bly from the presence of radioactivity mainly coinciding with the spots of the stained proteins.

Diagrams of [~4C]radioactivity distribution among the proteins isolated from the ribosomes labelled at different states are shown in Figs. 2-5. Each diagram represents t he average of three independent experiments. Differences in radioactivity of the same labelled proteins i n the experiments reproduced did not exceed 30% of the corresponding average values.

Environment of aminoacyl-tRNA at the A site As seen from Fig. 2, the sets of modified pi'oteins of

both subunits differ drastically for complexes (3)-(5) (Table III). Only two proteins, $5 and $7, are corm-non to all the three states. Proteins $8, S16. 317; LI4, L15

, and L24 are labelled exclusively in complex (3); $9, Sl l , S14, SI8, S19 and L21, L29 in (4); $2, L l l and L23 in (5), L9 is common for the states (3) and (4), and $21 for (4) and (5). These data indicate considerable changes in the protein environment of tRNA when passing both from complex (3) to (4) (i.e., as a result of GTP hydrolysis and EF-Tu removal) and from complex (4) to (5) (as a result of transpeptidation).

The P site in a factor-dependent system The sets of 30 S proteins labelled with (Phe),-azido-

tRNA P~ in post-translocation complex (9) (Fig. 4) and with az ido- tRNA~ in pretranslocation complex t~l) (Fig. 4) are less different than in the case of different states of the A site. Most of the protein sets - S5, Sl l , S13 and S 1 9 - are labelled in both cases, $9 and $21 only in post-translocation complex (9), and ST, S12 and S14 only in pretransiocation complex (11). The difference in the sets of labelled 50 ,'7, proteins is apprecia- ble in pre- and post, translocation complexes. In the post-translocation state only two proteins L17 and L27 are:modified, while in the pretranslocation state six proteins, L27 included. As was shown earlier by direcl photocrosslinking, the set of 30 S proteins crosslinked to :tRNA at the P site remains almost unchanged when passing from pre, to posttranslocation state (constant proteins $5; $ 9 / S l l , S13/S14/$27) in contrast to the set 0 fS0S proteins [4]. The set of 30 S proteins reported in: Ref. 4 is evidently included in the corresponding sets labelled with azido-tRNA.

7he P site in a non-enzymatic system The simplest and most common model systems are

non-enzymatically produced tRNA (deacylated or containing the analog of the peptidyl residue, N- acetylamino acid) complexes with ribosomes and templates. In the absence of at. excess of tRNA the binding occurs at the P site in such systems [13.14,18,19,30]. A set of proteins modified with deacylated azido-tRNA ~ in such complex (6)is shown in Fig. 3. A very similar set of proteins is produced when the complex (10) (Fig. 4) is obtained by treating the post-translocation complex with puromycin. The absence of S5 and LI4 in this case is rather due to a multistep selection of azido-tRNA molecules at the elongation steps, as a result of which the final complex does not contain azido-tRNA molecules with aryi azide groups on the residues modifying S5 and L14 in non-enzymatic complexes. Hence, an important conclusion can be drawn: the environment of tRNA at the P site of ribosome is independent of the way of the complex formation and depends on the nature of the final complex exclusively. Thus. such a non-enzymatic binding of tRNA does not seem incor- rect in terms of the structure of the forming complex obtained with the use of elongation factors.

Comparison of the data eb,~i~ed with azido4RNA-I and -1I

To obtain azido-tRNAl'hc-|, tRNA mc was alkylated with CE~CHzNH 2 under conditions of tRNA tertiary structure lability. As a result, the reactive groups were scattered almost statistically over atoms N-7 of guanine residt~es (with ;he exception of G27_3o. Gr~_ 77. and G~) and over s4U~ and X4~ [12,29]. Azido-tRNA-II was obtained under conditions of tRNA structure stability. The residues, whose nucleophilic sites were involved in the formation of the tRNA tertiary structure (G~5, G~, G53, G~7 , G63 , Grs , s4Ua and X47) remained unmodified [12,29]. By comparing the sets of proteins modified with different types of azido-tRNA P~ in identical complexes, (6) and (7) (Fig. 4), one can see that the set modified with azido-tRNA-ll is narrower than that of the derivative I. A wider set of the latter may be due to a greater number of distribution sites of the reactive groups in tRNA. The whole set of the proteins modified with azido-tRNA-ll is included in the corresponding set for azido-tRNA-l. Thus, aryl azide groups on the tRNAP% residues important for its native structure folding seem to be in contact with the ribosome. This suggestion is in agreement with numerous data indicating that the tRNA structure is either cha,~ed or somewhat melted i~ ribosome [311:

The situation is more complicated for the 70 S initiation complex (Fig. 5), in which the set of proteins modified with azido-tRNAtMCt-II is narrower than that for I but is not included in it completely ($7 and L27). This fact can be due to a more strict selection of the

; i 5 4 : : : . . . . . : '

modified tRNA~ et molecules at the steps of initiation (aminoacylation, binding to IF-2. GDP and to 30 S Subunits) as compared with tRNA ph~ at the elongation Steps. Probably the Set of fMet-azido-tRNAMet-I molecules in the 70 S initiation complex does not include the molecules bearing aryl azide groups at the positions responsible for= the modification of $7 and L27 in the case of functionally active azido-tRNAMe'=II (Tables I and i I ) : :

70 S initiation complex - 'I" site T h e sets of proteins modified with fMet-tRNA~ et

derivatives differ significantly from those for the tRNA Phe analogs at the P site at different states. For example, S1, $3, L1 and L7/L12 are modified with tRNA ph¢ derivatives at:neither of the functional sites. This phenomenon can be associated with boih a considerably different environment of tRNA~ et in the 'initiation' ('I') Site from that in the 'elongation' P si~e and with different spatial and primary structures of elol~ga- tor and initiator tRNA, The literature lacks data on environment of tRNA molecule in 70 S initiation complex. Using direct photocrosslinking it has been possible to observe only slight differences in the sets or ;::oteins crosslinked to fMet-tRNAr~ e~ and AcPhe-tRNA Ph¢ in ihe ¢O~aesponding Complexes with 30 S subunits and related templates [2,3]. However, these data arenot in Conflict wi th ours, as different complexes have been employed (30 S in Refs. 2, 3 and 70 S in the present Study) and :the approaches provide different information. Our results vividly show a difference in the structural arrangements o f ' I ' and 'P' sites as well as invalid- ity of application of AcPhe-tRNA for the modelling of initiation complexes.

Discussion

The reactive groups in azido-tRNA are flexible and have several rotation centers, their maximum length being 13 A in the elongated state with non-distorted valence angles. Thus, the modification with azido-tRNA gives information about the environment of the derivative within a radLts of up to 13 A of the tRNA frameworkl For direct crosslinking to occur, the tRNA base must be in close (less than 4 A) contact with the protein amino-acid residue, a definite mutual orientation of the crosslinking groups being indispensable [4]. Thus, both the approaches have limitations. The affinity

labelling approach provides more detailed information about tRNA environmen t and, consequently, Should be more sensitive t ° the possible alterations of the latter. However; in:large sets of proieins crosslinked to azido- tRNA, there can be those which do not interact directly with tRNA. Direct photocrosslinking gives information 0nly on the real contacts, but some of these contacts may: remain undetected due to stereochemical limita-

tions. So, the sets of proteins crosslinked directly to tRNA may not be included in the sets modified with azido-tRNA in a similar complex. Obviously, well- grounded conclusions can be made only from a comparison of the data obtained by the two methods.

Earlier, the sets of proteins crosslinked to Phe- tRNA phe in complexes (3) and (5) were shown to be considerably different by means of direct pilotocr::~s- linking [4]. It should be noted, however, t~,~ the sets obtained in Ref. 4 are not predominantly included in the corresponding sets obtained in the present study for azido-tRNA, which is quite reasonable (see the above speculations). The: study of the intermediate state (4) has not been included in Ref. 4; therefore, the differences in the sets of proteins crosslinked to t R N A at the A site before GTP hydrolysis and at the A site of the pretranslocation complex have been interpreted in two ways: by a different tRNA environment in these functional states and/or by the shielding: effect of the Tu factor in the type (3) complex. Our data suggest tha t considerable changes o f the protein : environment of tRNA at the A site also occur due tb the transpeptidation after EF-Tu has left the ribosome.

Protein $5 and $7 common to all the, three types of complex containing azido-tRNA in the A site may be reasnr~hlv assigned to the decoding region o f the A site. Location of $5 and S 7 i n Y,side of the decoding center is in agreement with the data on the affinity labelling of these proteins by oligoribonucleotides bearing a reactive group at the 3'-end (mRNA analogs) I in complexes with the ribosome and tRNA [32,33]. $5 was the only common protein wh:ch was crosslinked to tRNA at the A site both prior to GTP hydrolysis and after transpeptidation in Ref. 4, It was also attributed to the decoding center by the authors of Ref.: 4,

The data obtained are important for the interpreta' tion of results of the studies on the EF-Tu-dependent binding of amino-acyl-tRNA at the ribosomal A site reported by different authors. In many studies the location of aminoacyl-tRNA at the A site has been real- ized in a simple system in which deacylated tRNA was at the P site [6,10,34,35]. This state most resembles complex (4). As was discussed above, the arrangeme~a of tRNA in complexes of type (14) differs drastically from that in other types of complex containing tRNA in the A site. So, the: data obtained i n the simplified system containing deacylated tRNA in the P site a n d aminoacyl-tRNA in the A site could be attributed a t least to the complex of type (4), but by no means to the 'A site' ingeneral. =

The environment of azido-tRNA in the P site com- plexe s is variable preferentially for 50 S proteins. There, fore, the translocation (complex (l t ) - , complex (9), Fig, 4) considerably reduces the labelled set; L27 which is known to belong to the region of peptidyltransferase center [36,37] is the only common 50 S protein in p r e ,

i

a n d post-translocation states. Therefore, these results can be reasonably explained by the facts that in a post-translocation state the acceptor stem of the tRNA molecule at the P site is rigidly fixed via a strong interaction of the peptidyl moiety with the ribosome in the "peptide channel' [381. Thus, the environment of this part of tRNA is largely dependent on the presence of the peptidyl moiety in tRNA.

The state corresponding to complexes (5) and (10) wi th deacylated tRNA at the P site an~ ~acant A site neve r occurs in the elongation cycle. I t is not surprising. therefore, that a set of modified proteins corresponding t o this simplified state is somewhat different from those in p re -and post-translocation states (11) and (9). How- eve r , most o f the proteins modified in the simplified state (6) or (10 )occur in the sets corresponding to complex (9) or (11). A simplified pretranslocation complex (8) I (Fig. 3), in which azido-tRNAPh~-ll is bound non-enzymatically in the P site and Phe-tRNA pn~ in the A site by EF-Tu and GTP, yields a set of modified proteins all of which (except of L33) are included in the set corresponding to the 'true" complex (in complex (8)

a set of modified proteins is reduced in comparisor, with complex ( l l ) i since in the former case azido-tRNA-ll with a smaller amount of sites bearing aryl azide groups has been: used: s ee Results). This can be taken as evidence for the 'correctness" of the structural arrangement o f the: P site in ~ the pretranslocation complex obta ined using non-enzymatic binding of tRNA at the P site. It should be noted that, according to Ref. 4, the structural organization of the A site in the pretranslocation complex obtained without EF-Tu is noticeably different from that of the 'correct' complex.

:Protein $5 is common for all states studied for azido-tRNA both in t h e A and P sites, the latter fact

be ing confirmed by the direct photocrosslinking [4]. So, this protein can be attributed not only to the 3'-side but also to 5'-side of the decoding region. Of the remainder three 'constant" proteins at the P site, namely S l l , S13. S I9 , that are modified with azido-tRNA not only in pre . and post-translocation states but: also upon the non-enzymatic binding at the P site (Fig. 3, complex (6)). S13 can be attributed on the most firm basis to the decoding region. Among the 30 $ proteins, S13 is the only :one whose modification under non-enzymatic binding of azido-tRNA e~ depends upon poly(U)[14]; in addition, an isolated S13 protein shows affinity for poly(U) which strongly increases: in the presence of tRNA p~ [391.

Azido-tRNAPh~,l has been recently applied to the affini:ty labelling of the E site of 70 S ribosomes [401,

which is an additional site of codon,independent binding of :the deacylated tRNA for the case ,vhen the P site i s blocked by AcPhe, tgNA or edeine [30,40]. At the E

i site which is supposed to be a n 'exit' one [30], a set of proteins $8, $15; $17, $18, $21 and L10 having nothing

z

155

• 6 - g

cl 13 c

Fig. 6. Three-dimensional models of the 30 S subunit of E, ¢oli. The numbers in the model indicate the locations of the center~ ~f antib~,dy binding sites for individual ribosomal proteins as given in Rcfs~ 4! (a, b: different views of 30 S model) and 42 (c). The arrO~ indicates the position of the protein S19 as determined in Ref. 43. Ligli~iy ,.,haded

sites shown in (c) are located on the far side of lhc ~ubuniL

in common with the sets corresponding to any tRNA state at A or P site has been modified. The set for the E site has no proteins attributed by an)one to the decoding region. This indicates that the E site is most probably remote from this region and physically separated from the A and P sites. rh~ ~ :ieins modified with azido-tRNA in any st:l!:: : form any compact domain commensurable ..... : ~RNA molecule on the existing models of 70 S :~:~omes [41,42]. Even if the groups of 30 S proteins crosslinkable to azido-tRNA in any state at the A or P site are considered, such domains will be still difficult to distinguish (Fig. 6). For most states, relatively small sets of the proteins contact- ing with tRNA (direct photocrosslinking data) also fail to form compact domains on the ribosome model f4] The :;:uthors [4] have attempted to explain this fact in two ways: (i) insufficiently accurate location of proteins on the models; and (ii) possible elongation of the L- shaped tRNA molecule due to the ~'.hange of its macrostructure in the riboson ~lhese reasonings do not seem convincing enough, sinc~ they cannot account for the multiplicity of the facts of tRNA crosslinking (in one state) to the proteins located on the surface of subunits at distances larger than the length of tRNA and even on different sides of the 30 S subunit.

As early as in 1968, a hypothesis was put forward implying that the structure of ribosome5 is not static during translation. Particularly, the mutual orientation of subunits (linking, up - opening)and their internal structure tend to change during: translocation [4,*]. This hypothesis has been confirmed later [45;46]. We propose a more general principle for the action of ribosomes: (i) formation o f each type of complex i s accompanied not only by possible changes i n the tRNA macrostructure but also by structural changes of ribosomal subunits themselves (SPeCifically, o f mutual orientation of pro, reins which spreads allostericatl~ from the 'starting' tRNA binding site - 'conformatioaa! adjustment' of the ribosome); ( i i ) the ribosome can change its internal structure dynamically not only durio~ translocation but

iii~i•::?!:: :? i•! • : / • • : • 156 ~ : :

also a t each step o f initiation and elongation; for this reason, the application of statL, tical models of ribo-

s o m e s is limited even if the ioc~lization of proteins is ideally accurate. This principle is additionally supported by the f ac t tha t the c o n t a c t s of t h e 16 S r R N A wi th

p r o t e i n s Change u p o n t R N A b i n d i n g , the c h a n g e s b e i n g

d e p e n d e n t even o n t he p r e s e n c e o f a n a m i n o a c y l r e s idue

o n t he Y - e n d o f t R N A [3].

Thus;: t he re a re n o r ig id ly f ixed s t r u c t u r e s f o r m i n g

each type o f t R N A - b i n d i n g s i tes ( p r i m a r i l y A a n d P

sites ) on the70 S ribosomes. In each particular state a unique structural arrangement is observed. It should be noted that different states of the P site, functional and simplified model ones, are more similar to each other than the states of the A site~

Acknowledgements

The authors are grateful to Drs S.V. Kirillov and Yu.P. Semenkov for helpful discussions.

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Documents

Structural arrangement of tRNA binding sites on Escherichia coli ribosomes, as revealed from data on affinity labelling with photoactivatable tRNA derivatives