Novel data on interactions of elongation factor Ts

Biochimie (1992) 74, 419-425 © Soci6t6 franqaise de biochimie et biologic mol6culaire / Elsevier, Paris

419

Novel data on interactions of elongation factor Ts MG Bubunenko, ML Kireeva*, AT Gudkov Institute of Protein Research, Russian Academy of Sciences, Pushchino 142292, Moscow Region, Russia (Received 26 August 1991; accepted 15 November 1991)

Summary m Interactions of EF-Ts with EF-Tu at all steps of the elongation cycle were studied by limited trypsinolysis, gel-filtration, analytical centrifugation and fluorescence polarization techniques. It is shown that EF-Ts does not dissociate from EF-Tu after GDP to GTP exchange, but remains bound to the Aa-tRNA~.EF-Tu,GTP complex up to GTP hydrolysis stage on the ribosome. The possible role of these interactions is discussed. elongation factor Ts / interaction with EF.Tu-Aa-tRNA complex / interaction with ribosome

Introduction Elongation factor EF-Ts is known to take part in the regeneration of the Aa-tRNA.EF-Tu.GTP complexes that are essential for binding of Aa-tRNA to the ribosome [1]. It is considered that EF-Ts accelerates the exchange of EF-Tu-bound GDP to GTP, catalyzing the release of G D P from EF-Tu [2]. The exchange reaction proceeds via the formation of kinetically labile complexes EF-Tu.GDP.EF-Ts and EF-Tu.GTP. EF-Ts [3]. The stimulation of G D P to G T P exchange is the only reliably known role of elongation factor Ts in protein biosynthesis [4]. However, there are data presuming that the role of EF-Ts may not be restricted to the catalysis of nucleotide exchange. For example, coordination of the amounts of EF-Ts and ribosomes at all growth rates [5] probably indicates that EF-Ts participates in ribosome-dependent steps of elongation. Some results of kinetic analysis of the exchange reaction also suggest that EF-Ts could participate in the following exchange reaction stages of EF-Tu cycle as the EF-Tu.GTP.EFTs complex is comparatively stable [3]. To elucidate new aspects of EF-Ts function we have analyzed interactions of EF-Ts with EF-Tu at all steps *Correspondence and reprints Abbreviations: EF-Tu, EF-Ts, elongation factors Tu and Ts; Aa-tRNA, aminoacylated tRNA; Phe-tRNAphe, phenylalanylspecific tRNA charged with phenylalanine; GMPPNP, guanylylimidodiphosphate, an uncleavable analog of GTP; DnsCl, dansylchloride; poly(U)-polyuridilic acid.

of the elongation cycle, from the formation of the complex with GTP to GTP hydrolysis on the ribosome. The results show that EF-Ts remains bound with EF-Tu up to GTP hydrolysis on the ribosome and, consequently, has an additional functional role in elongation of the polypeptide chain. Materials and m e t h o d s

Enzymes, chemicals and preparations GDP, GMPPNP and phosphoenolpyruvate were purchased from Serva, Heidelberg; GTP and tRNAabe were from Boehringer-Mannheim; trypsin (EC 3.4.21.5) and dansylchloride were from Sigma, USA; [3HIGDP, [32P]GTP, [t4Clphenylalanine and [3H]leucine were from Amersham International, UK; tRNA was from Biolar, Russia. The antibiotic kirromycin was kindly provided by Dr A Liljas, Lund, Sweden. 70S ribosomes were obtained as in [6]. Subunit association was approximately 90% as determined by analytical centrifugation. Aminoacylation and isolation of tRNA were performed as described in [7]. EF-Tu.GDP.EF-Tu.Ts and EF-Ts were isolated as described in [8]. The functional properties of EF-Tu were checked as in [9] and the properties of EF-Ts were analyzed by GDP exchange stimulation assay method described in [ 101. The factors were found to be 90-95% active in all activity assays. The following buffer was used in all experiments: 40 mM Tris-HCl, pH 7.6, 10 mM MgCI2, 50 mM NH4CI, 1-2 mM dithiothreitol. Analytical gel-filtration Analytical gel-filtration [ 11] was used to study the interaction of EF-Tu.Ts with GDP and GTE The sample (40 ~!) containing 40 nmol EF-Tu.Ts and 2 nmol [3H]GDP or )'-[32p]GTP was

420 applied to a column with Ultrogel AcA-44 (0.25 x 30 cm) and eluted with the buffer containing 2 I~M nucleotide. The 45-t.d fractions were analyzed by SDS-electrophoresis, total radioactivity estimation or by GDP exchange of EF-Tu [ 10].

Limited trypsinolysis of EF-Ts and ribosomes The formation of factor-ribosome complexes was carried out by 10 min incubation at 37°C in a mixture containing 6 ~M ribosomes charged with poly(U) and tRNA [12], and other components: 5 I~M EF-Tu,Ts, 8 ~tM Phe-tRNA Phe, 1 mM nucleotide (GTP or GMPPNP) and 100 ~tM kirromycin when required. Analysis of EF-Ts stability to proteolysis was carried out by trypsin treatment. Trypsin concentration was 3 ~g/ml. The mixture was incubated at 37°C, the aliquots were withdrawn at defined time intervals and analyzed by SDS-electrophoresis [13] as described in [14], Trypsinolysis of ribosomes was carried out as described previously [9]. The reaction mixture contained 6 llM of charged ribosomes and a three-fold excess of the complexes of EF-Tu with Aa-tRNA. The trypsin/total protein ratio was 1:500 and the incubation was performed for 15 rain at 37°C. The ribosomal proteins were extracted and analyzed by two-dimensional gel-electrophoresis [ 15].

Analytical centrifugation The factor-ribosome complexes used in the experiment were prepared as described above with the exception that the reaction mixture contained 6 I~M EF-Tu.Ts and 10 pM charged ribosomes. The samples (150 pl) were subjected to centrifugation for 2.5 h at 28 000 rpm in a SW-55 rotor. The aliquots of the supematant were immediately withdrawn after centrifugation and the fractions were analyzed by SDS-electrophoresis [ 13] followed by scanning of the Coomassie blue-stained gel.

Fluorescence polarization measurements To avoid inactivation of EF-Ts in the process of labeling the dansyl derivative of EF-Ts was obtained by treating the purified EF-Tu.Ts complex with dansylchloride followed by dissociation of the labeled complex to EF-Tu.GDP and EF-I~ according to [8]. The EF-Tu.Ts to dansylchloride molar ratio was 1:200. The reaction was preformed in buffer 40 mM Tricin-NaOH, pH 8.5, 3 mM mercaptoethanoi for 12 h at 4°C. A standard procedure [8] was used to obtain the EF-Tu.(DnsEF-Ts) complex. The complexes of EF-Tu with various ligands were prepared as described above. Samples (1 ml) contained 20 14M GDP or GTP with a GTP-regeneration system [7] or 100 I~M GMPPNP and, when required, equimolar quantities of EF-Tu.GDP and Phe-tRNA Phe and a three-fold excess of

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the ribosomes charged with poly(U) and tRNA. The stability of interaction of Aa-tRNA with EF-Tu in the absence of ribosomes was determined by protection of Aa-tRNA from non-enzymatic deacylation in Tris buffer [ 16]. Fluorescence measurements were performed in a 1.0-cm quartz cuvette at 25°C on an Aminco SPF-600 spectrofluorimeter supplied with a polarizer. The excitation monochromator was set at 340 run and fluorescence intensities were detected at 545 nm. The polarization values were determined according to [17, 18] using the scatter-corrected intensities. The scattering levels were determined for each sample before addition of the dansyl-labeled factor. The exactness of the polarization value determination was about 0.002--0.003 arbitrary units.

GTP.EF-Ts complexes are not just kinetic intermediates of the exchange reaction, but are quite stable complexes that exist even in the presence of nucleotide excess and can be assayed by various methods. On the one hand, when EF-Tu.Ts interacts with tritium-labeled G D P the position of the protein-bound nucleotide peak eluted from the gel-filtration column equilibrated with [3H]GDP coincides with that of the EF-Tu.Ts but not with the EF-Tu.GDP peak (fig 1). EF-Ts interacts with EF-Tu GTP in the same way (data not shown). Furthermore, GTP and G D P change the susceptibility of factor EF-Ts in the EF-Tu.Ts complex to trypsinolysis. It can be seen (fig 2) that the cleavage rate of EF-Ts increases in the presence of G T P and, to a greater extent, in the presence of G D P in comparison with that of EF-Ts in the EF-Tu.Ts complex without nucleotides, but remains less than the cleavage rate of free EF-Ts. These results demonstrate that EF-Tu.Ts could form quite stable complexes with G D P and GTP. On the other hand, we analyzed the interaction of dansyl derivative of EF-Ts with the preformed EFTu.GDP and EF-Tu.GTP complexes. Within the used concentration range only EF-Tu.GTP and not EFTu.GDP causes the increase of fluorescence polarization value (fig 3). This result indicates that the affinity of the factor EF-Ts to EF-Tu.GTP is higher than to EF-Tu.GDP. It is necessary to note that if the catalysis of the nucleotide exchange is the only EF-Ts function it might be supposed that the EF-Ts affinity to the sub-

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Cell-free translation system of poly( U) The rate of polyphenylalanine synthesis was estimated as described in [7] with a modification: the isolated ternary complexes were used as a source of phenylalanine. The isolation of Phe-tRNA.EF-Tu.GTP complexes was performed by gel-filtration on Ultrogel AcA-44. The obtained ternary complexes were pre-incubated with EF-Ts and added to the remaining components of the translation system. Translation accuracy is expressed as the ratio of incorporated phenylalanine and leucine and was measured according to [ 19]. In this case ternary complexes were also pre-formed before addition of Aa-tRNA and EF-Tu to the reaction mixture.

Results and discussion Analysis of EF-Tu interactions with EF-Ts, G D P and G T P demonstrates that EF-Tu.GDP.EF-Ts and EF-Tu.

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strate of the reaction, EF-Tu.GDP, would be higher than to EF-Tu.GTP. However, the situation is reversed and therefore EF-Ts may participate in the subsequent reactions with factor EF-Tu, particularly in the formation of the complex with Aa-tRNA. Indeed, it was shown that EF-Ts interacts with the ternary complexes, as Aa-tRNA.EF-Tu.GTP increases dansyl-EF-Ts fluorescence polarization value. It is essential that the ternary complexes induce a higher increase of the polarization value than does the same concentration of EF-Tu.GTP (fig 4). This indicates a higher affinity of EF-Ts to Aa-tRNA.EF-Tu.GTP than to EF-Tu.GTP. Thus, the obtained data demonstrate that EF-Ts interacts with EF-Tu.GTP after the nucleotide exchange and can participate in the formation of the complex with Aa-tRNA. High EF-Ts affinity to Aa-tRNA.EFTu.GTP complex probably indicates that EF-Ts together with this complex binds to the riboso~e. This assumption has been checked in various experiments. Analysis of the EF-Ts-EF-Tu interactions was carried out on factor-ribosome complexes modelling two states of EF-Tu on the ribosome. The pre-hydrolysis state is achieved by using the non-hydrolizable analog of GTP and the post-hydrolysis state by adding the antibiotic kirromycin which increases the affinity of EF-Tu.GDP to the Aa-tRNA and the ribosome. The following data demonstrate the EF-Ts interaction with EF-Tu on the ribosome. l) Fluorescence polarization value of dansyl-EF-Ts in the presence of Tu-ribosome complex with GMPPNP

is higher than that in the presence of ribosomes without EF-Tu. At the same time the addition of the Aa-tRNA.EF-Tu.GDP.kirromycin complexes does not affect the polarization (fig 5). 2) The ratio of EF-Tu and EF-Ts amounts in supernatant fraction after centrifugation of EF-Tu.Ts with various factor-ribosome complexes depends on the type of the complex used in the experiment. The data given in table I indicate that EF-Ts cosediments with the ribosomes in pre-hydrolysis state and does not sediment when the ribosomes are in post-hydrolysis state. 3) The results of trypsinolysis show that EF-Ts changes the susceptibility of some ribosomal proteins to the cleavage. In the complexes of ribosomes with Aa-tRNA,EF-Tu.GMPPCP.EF-Ts leads to the additional cleavage of the protein L l 9 whereas protein L27, on the contrary, becomes resistant to cleavage (table II). The sensitivity of EF-Ts to proteolysis depends on the type of the EF-Tu-ribosome complex. As seen in figure 6, EF-Ts is more stable in the presence of the EF-Tu-ribosome complex in the prehydrolysis state. The cleavage time of EF-Ts with the ribosomes in the post-hydrolysis state is equal (data not shown). The data summarised above indicate that EF-Ts with Aa-tRNA.EF-Tu.GTP binds to the ribosome and

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423 Table I. Content of EF-Tu and EF-Ts in the supernatant fraction (arbitrary units after gel scanning). EF-Tu

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that dissociation could occur after GTP hydrolysis. Moreover, the results of trypsinolysis show that EF-Ts most likely influences the mode of interaction of the Aa-tRNA and EF-Tu with the ribosome. Thus, EF-Ts was demonstrated to interact with EFTu not only in the nucleotide exchange reaction but also in the subsequent stages of the elongation cycle. This result supports the assumption that the catalysis of nucleotide exchange is not the only function of factor EF-Ts. Various possible functions of EF-Ts not related to nueleotide exchange can be presumed. Firstly, EF-Ts interacts with Aa-tRNA.EF-Tu.GTP before the binding of the Aa-tRNA to the ribosome. We think that the comparatively tight binding of EFTs to the ternary complexes could decrease the amount of EF-Ts able to catalyze the exchange reaction. This idea has been checked in in vitro systems of nucleotide exchange. It was shown (table III) that preformed ternary complexes significantly decrease EF-Ts-dependent nucleotide exchange. It means that if the catalysis of the exchange reaction is an essential prerequisite for ternary complexes formation, then the Aa-tRNA. EF-Tu.GTP regeneration rate should be inhibited by ternary complexes themselves. In this case the amount of ternary complexes participating in protein synthesis should be similiar to the amount of EF-Ts and also of

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Fig 6. SDS-electrophoresis of EF-Tu.Ts complexes treated with trypsin in the presence of ribosomes. The samples were taken after 2.5, 5, 10 and 20 rain trypsin treatment. Lane 1: ribosomes + native EF-Tu.Ts; lanes 2-5: ribosomes. poly(U).tRNA + Aa-tRNA; EF-Tu.Ts, GMPPNP; lanes 6-9: ribosomes.poly(U).tRNA + Aa-tRNA, EF-Tu.Ts, GTP, kirromycin.

ribosomes since content of EF-Ts in the cell is identical to the content of ribosomes at all growth rates [5, 20]. It is quite important as the excess of AatRNA.EF-Tu.GTP complexes over the ribosomes influences translational accuracy [21]. Therefore, the coordination of ternary complexes and ribosomes amounts via EF-Ts could be essential for maintaining definite rate and accuracy of protein synthesis. Secondly, EF-Ts together with EF-Tu binds to the ribosome. We believe that EF-Tu conformation in the Table HI. Effect of ternary comp!exes on EF-Ts-dependent GDP/[3H]GDP exchange rate.

Table II. Proteins digested by trypsin in 70S ribosomes in the presence of elongation factors EF-Tu and EF-Ts.

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Fig 7. Effect of EF-Ts on the rate and accuracy of poly(U) translation. (a) Rate of polyphenylalanyl synthesis without EF-Ts (1) and with 500 pmol EF-Ts (2). The reaction mixture contained 80 pmol Phe-tRNAPhe.EF-Tu.GTP.(b) The phenylalanine to leucine ratio without EF-Ts (1) and in the presence of 2 nmol EF-Ts (2). The reaction mixture contained 240 pmol PhetRNAPhe.EF-Tu.GTPand 240 pmol Leu-tRNAPhe.EF-Tu.GTP.

quaternary complex Aa-tRNA.EF-Tu.EF-Ts.GTP differs from EF-Tu conformation in the complex without EFTs. Consequently, EF-Ts should affect EF-Tu interactions both with ribosomes and with Aa-tRNA. On the one hand, the interaction of EF-Ts with EF-Tu in the process of Aa-tRNA binding to the ribosome could enlarge size and asymmetry of the bound particle. The appearance of an additional contact site due to EF-Ts would probably increase the specificity of the interaction. Moreover, taking into consideration the similarity between the EF-Tu conformational changes induced by EF-Ts and kirromycin [22, 23], we can assume that EF-Ts together with the ribosome provides the requisite conformation of the EF-Tu-AatRNA complex that is essential for GTP hydrolysis, as is kirromycin [24]. Thus, it is quite possible that EFTs directly or indirectly affects the interaction of EFTu and Aa-tRNA with the ribosome. On the other hand, if EF-Ts binds to the ribosome it could be supposed that all the reactions requiring elongation factors Tu and Ts, including the GDP to GTP exchange, occur near the ribosome and this could increase the efficiency of protein biosynthesis. We have made an attempt to check EF-Ts role in translation system when the requirement for GDP to GTP exchange was excluded by the use of ready-

made ternary complexes. But in a wide range of EFTs concentration only a slight stimulation of the poiy(Phe) synthesis was observed (fig 7a) in agreement with the previous data [25]. The accuracy of translation did not depend on EF-Ts (fig 7b). However, we assume that the standard cell-free system used in the experiments markedly differs from the intra-cellular conditions by several important parameters: mRNA composition and structure, concentration of factors, nucleotides and, especially, the total concentration of macromolecules. EF-Ts probably could have a noticeable influence on the ribosome-dependent steps of the elongation cycle in vivo where the high concentration of macromolecules favors stabilization of the weak complexes [26]. Thus, we have demonstrated that EF-Ts interacts with EF-Tu not only in the course of nucleotide exchange, but also during formation of the complex with Aa-tRNA and its binding to the ribosome. We believe that these interactions indicate a novel function of factor EF-Ts in protein biosynthesis.

Acknowledgments The authors thank Prof AS Spirin for support of this work and Dr TL Bushueva for help in fluorescence studies.

425

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