The Elongation Factor 3 Unique in Higher Fungi and Essential for Protein Biosynthesis Is an E Site Factor
1995; Elsevier BV; Volume: 270; Issue: 35 Linguagem: Inglês
10.1074/jbc.270.35.20473
ISSN1083-351X
AutoresFrancisco J. Triana‐Alonso, Kalpana Chakraburtty, Knud H. Nierhaus,
Tópico(s)Peptidase Inhibition and Analysis
ResumoTwo elongation factors drive the ribosomal elongation cycle; elongation factor 1α (EF-1α) mediates the binding of an aminoacyl-tRNA to the ribosomal A site, whereas elongation factor 2 (EF-2) catalyzes the translocation reaction. Ribosomes from yeast and other higher fungi require a third elongation factor (EF-3) which is essential for the elongation process, but the step affected by EF-3 has not yet been identified. Here we demonstrate that the first and the third tRNA binding site (A and E sites, respectively) of yeast ribosomes are reciprocally linked; if the A site is occupied the E site has lost its binding capability, and vice versa, if the E site is occupied the A site has a low affinity for tRNAs. EF-3 is essential for EF-1α-dependent A site binding of aminoacyl-tRNA only when the E site is occupied with a deacylated tRNA. The ATP-dependent activity of EF-3 is required for the release of deacylated tRNA from the E site during A site occupation. Two elongation factors drive the ribosomal elongation cycle; elongation factor 1α (EF-1α) mediates the binding of an aminoacyl-tRNA to the ribosomal A site, whereas elongation factor 2 (EF-2) catalyzes the translocation reaction. Ribosomes from yeast and other higher fungi require a third elongation factor (EF-3) which is essential for the elongation process, but the step affected by EF-3 has not yet been identified. Here we demonstrate that the first and the third tRNA binding site (A and E sites, respectively) of yeast ribosomes are reciprocally linked; if the A site is occupied the E site has lost its binding capability, and vice versa, if the E site is occupied the A site has a low affinity for tRNAs. EF-3 is essential for EF-1α-dependent A site binding of aminoacyl-tRNA only when the E site is occupied with a deacylated tRNA. The ATP-dependent activity of EF-3 is required for the release of deacylated tRNA from the E site during A site occupation. INTRODUCTIONRibosomes from organisms of all kingdoms require two factors in the course of the reactions of the elongation cycle; elongation factor 1α (EF-1α) 1The abbreviations used are: EFelongation factorAMP-PNPadenosine 5′-(β,γ-imino)triphosphate. is responsible for the codon-dependent selection of the cognate aminoacyl-tRNA at the ribosomal A site and elongation factor 2 (EF-2) for the translocation reaction. The discovery of a third elongation factor (EF-3) essential for the elongation cycle in the yeast Saccharomyces cerevisiae was therefore a surprise(1Skogerson L. Wakatama E. Proc. Nat. Acad. Sci. U. S. A. 1976; 37: 73-76Crossref Scopus (60) Google Scholar). Furthermore, this factor is a ribosome-dependent ATPase, which also accepts GTP and ITP as substrates and to a lesser extent pyrimidine nucleotides(2Dasmahapatra B. Chakraburtty K. J. Biol. Chem. 1981; 256: 9999-10004Abstract Full Text PDF PubMed Google Scholar, 3Uritani M. Miyazaki M. J. Biochem. (Tokyo). 1988; 103: 522-530Crossref PubMed Scopus (42) Google Scholar, 4Kamath A. Chakraburtty K. J. Biol. Chem. 1989; 264: 15423-15428Abstract Full Text PDF PubMed Google Scholar). This wide substrate specificity of EF-3 is one principal difference from other translational factors, which show a stringent requirement for GTP(5Moldave K. Annu. Rev. Biochem. 1985; 54: 1109-1149Crossref PubMed Scopus (357) Google Scholar).EF-3 from S. cerevisiae consists of a single polypeptide chain of 1044 amino acids(6Chakraburtty K. Fernandes P. New Approaches to Anti-fungal Agents. Birkhäuser Verlag, Basel1992: 114-142Google Scholar, 7Sandbaken M. Lupisella J.A. DiDomenico B. Chakraburtty K. J. Biol. Chem. 1990; 265: 15838-15844Abstract Full Text PDF PubMed Google Scholar). The genes encoding the EF-3 analogues in Candida albicans and in Pneumocystis carinii, higher fungi than yeast, have also recently been isolated and sequenced (8Qin S. Xie A. Banato C.M. McLaughlin C. J. Biol. Chem. 1990; 265: 1903-1912Abstract Full Text PDF PubMed Google Scholar, 9DiDomenico B.J. Lupisella J. Sandbaken M. Chakraburtty K. Yeast. 1992; 8: 337-352Crossref PubMed Scopus (36) Google Scholar, 10Ypma-Wong M. Fonzi W. Sypherd P.S. Infect. Immun. 1992; 60: 4140-4145Crossref PubMed Google Scholar). The deduced amino acid sequences from these two species show over 75% identity with the EF-3 from S. cerevisiae. The Candida EF-3 gene could supplement the disrupted EF-3 gene in S. cerevisiae, demonstrating the functional identity of EF-3 among different fungi(9DiDomenico B.J. Lupisella J. Sandbaken M. Chakraburtty K. Yeast. 1992; 8: 337-352Crossref PubMed Scopus (36) Google Scholar). The activity of EF-3 is distinct from that of EF-1α and EF-2, since EF-1α and EF-2 from yeast can functionally replace their counterparts in the presence of ribosomes from rat liver or wheat germ, whereas an EF-3 dependence is only observed with yeast ribosomes(11Skogerson L. Engelhardt D. J. Biol. Chem. 1977; 252: 1471-1475Abstract Full Text PDF PubMed Google Scholar, 12Chakraburtty K. Kamath A. Int. J. Biochem. 1988; 20: 581-590Crossref PubMed Scopus (29) Google Scholar). This dependence follows the source of 40 S subunits as suggested by experiments with hybrid ribosomes(12Chakraburtty K. Kamath A. Int. J. Biochem. 1988; 20: 581-590Crossref PubMed Scopus (29) Google Scholar). A monoclonal antibody against EF-3 blocks in vitro translation of poly(U) as well as natural mRNA, and evidence was obtained that EF-3 was associated with polysomes rather than with ribosomal subunits and that the protein is required for every cycle of the elongation of the polypeptide chain(13Hutchinson J.S. Feinberg B. Rotwell T.C. Moldave K. Biochemistry. 1984; 23: 3055-3063Crossref PubMed Scopus (25) Google Scholar). Polyclonal antibodies did not cross-react with extracts from mammals (liver and reticulocytes) or plants (wheat germ) and, accordingly, did not affect the respective translational systems(12Chakraburtty K. Kamath A. Int. J. Biochem. 1988; 20: 581-590Crossref PubMed Scopus (29) Google Scholar).The actual step of the elongation cycle promoted by EF-3 has not been identified. The factor is not involved in the nucleotide exchange reactions of either EF-1α or EF-2(4Kamath A. Chakraburtty K. J. Biol. Chem. 1989; 264: 15423-15428Abstract Full Text PDF PubMed Google Scholar). Slight stimulations of some other partial reactions of the elongation cycle including peptide bond formation and translocation have been reported(14Miyazaki M. Uritani M. Kagiyama H. J. Biochem. (Tokio). 1988; 104: 445-450Crossref PubMed Scopus (17) Google Scholar, 15Miyazaki M. Kagiyama H. J. Biochem (Tokyo). 1990; 108: 1001-1008Crossref PubMed Scopus (17) Google Scholar). More detailed examinations revealed that aminoacyl-tRNA binding to the A site was stimulated by EF-3 but only in the presence of catalytic amounts of EF-1α(4Kamath A. Chakraburtty K. J. Biol. Chem. 1989; 264: 15423-15428Abstract Full Text PDF PubMed Google Scholar), which does not easily explain the absolute requirement found for EF-3 in vivo(7Sandbaken M. Lupisella J.A. DiDomenico B. Chakraburtty K. J. Biol. Chem. 1990; 265: 15838-15844Abstract Full Text PDF PubMed Google Scholar).EXPERIMENTAL PROCEDURESTransfer RNA IsolationFor the isolation of specific tRNAs from yeast a new method was been used, 2F. Triana-Alonso and K. H. Nierhaus, unpublished observations. which followed the following principle. Bulk tRNA from yeast was charged with the radioactively labeled amino acid corresponding to the tRNA to be isolated and then acetylated at the α-amino group, which stabilized the 3′-ester bond for the subsequent high performance liquid chromatography separation. Fractions containing N-acetyl-aminoacyl-tRNA were enzymatically deacylated by the peptidyl-tRNA hydrolase isolated from Escherichia coli and then rechromatographed, yielding purified tRNAs with a specific amino acid acceptance of better than 1600 pmol/A260 unit. When necessary the isoaccepting tRNAs could be separated on a Bioselect 300-5 C4 using a gradient made from solution A (20 mM NH4OAc, pH 5.5, 5 mM MgCl2, 1000 mM sodium formate) and solution B (20 mM NH4OAc, pH 5.5, 10% MeOH) described by Xue et al.(16Xue J. Shen W. Wong J.T. J. Chromatogr. 1993; 613: 245-247Crossref Scopus (16) Google Scholar).Transfer RNA Binding to RibosomesIt was important to observe the following technical points in order to obtain reproducible data in the tRNA-binding experiments: 1) The 80 S ribosomes must be poor in RNases. 80 S ribosomes were isolated and pelleted through a glycerol cushion in the presence of 0.5 M KCl as described (2Dasmahapatra B. Chakraburtty K. J. Biol. Chem. 1981; 256: 9999-10004Abstract Full Text PDF PubMed Google Scholar) and then treated with puromycin (1 mM) and GTP (1 mM)(17Martin T.E. Wool I.G. Castles J.J. Methods Enzymol. 1971; 20: 417-429Crossref Scopus (31) Google Scholar). 2) The [32P]tRNA should be purified via a sequencing gel before the isotope is diluted with nonlabeled tRNA as described previously(18Gnirke A. Geigenmüller U. Rheinberger H.-J. Nierhaus K.H. J. Biol. Chem. 1989; 264: 7291-7301Abstract Full Text PDF PubMed Google Scholar). 3) The AcPhe-tRNA should be enzymatically freed of contaminating Phe-tRNA. The residual Phe-tRNA present forms AcPhe-(Phe)n-tRNA on poly(U) programmed ribosomes, which mimic a higher value for AcPhe-tRNA binding. The Phe-tRNA contamination can be reliably removed by reversal of the enzymatic aminoacylation. For this purpose standard AcPhe-tRNA preparations were incubated in the presence of S-100 enzymes freed of tRNA, 4 mM pyrophosphate, and 4 mM AMP for 5 min at 30°C. Other parameters important for reliable values for AcPhe-tRNA binding are as follows. 1) "Tight coupled" ribosomes should be used, which are genuine "run-off" ribosomes according to Hapke and Noll(19Hapke B. Noll H. J. Mol. Biol. 1976; 105: 97-109Crossref PubMed Scopus (72) Google Scholar). In vitro reassociated ribosomes sometimes erroneously termed "tight coupled" ribosomes bind more than one AcPhe-tRNA per ribosome. 2) The specific activity for the [14C]Phe given by Amersham Corp. can be too high by up to 30% and must be redetermined for each batch. 3F. J. Triana-Alonso, C. Spahn, N. Burkhardt, B. Röhrdanz, and K. H. Nierhaus, unpublished observations. The elongation factors EF-1α, EF-2, and EF-3 from yeast were purified as described (2Dasmahapatra B. Chakraburtty K. J. Biol. Chem. 1981; 256: 9999-10004Abstract Full Text PDF PubMed Google Scholar, 20Dasmahapatra B. Skogerson L. Chakraburtty K. J. Biol. Chem. 1981; 256: 10005-10011Abstract Full Text PDF PubMed Google Scholar, 21Kamath A. Chakraburtty K. J. Biol. Chem. 1986; 261: 12596-12598Abstract Full Text PDF PubMed Google Scholar) and were homogeneous according to acrylamide gel electrophoresis (Fig. 1).Synthesis of Model Heteropolymeric mRNAsThe MF-mRNA was synthesized using a modified run-off in vitro transcription system with T7 RNA polymerase, which improves the accuracy of the synthesis of small RNAs with low content of secondary structure. 4F. Triana-Alonso, M. Dabrowski, J. Wadzack, and K. H. Nierhaus, unpublished observations. A typical transcription assay was performed as follows. 50-100 μg of linearized plasmid DNA template (final concentration of 20 pmol/ml) were incubated for 3 h at 37°C in a transcription mix containing 40 mM Tris-HCl, pH 8.0 (37°C), 22 mM MgCl2, 1 mM spermidine, 5 mM dithioerythritol, 100 μg/ml of bovine serum albumin (RNase- and DNase-free), 1000 units/ml ribonuclease inhibitor (RNasin, Promega, Madison, WI), 5 units/ml of inorganic pyrophosphatase, 3.75 mM each of ATP, GTP, and CTP, 0.9 mM UTP (a lower UTP concentration with respect to the other NTPs is essential for the improvement in accuracy), and 40 μg/ml of purified T7 RNA polymerase. After this incubation the mix was phenol-extracted, and the transcript was purified by polyacrylamide gel electrophoresis under denaturing conditions. The MVF-mRNA was chemically synthesized in an Applied Biosystems 392 RNA/DNA synthesizer using RNA phosphoramidites from Waters, Millipore, following a synthesis and deprotection protocol as described by Scaringe et al.(22Scaringe S.A. Franklyn C. Usman N. Nucleic Acids Res. 1990; 18: 5433-5441Crossref PubMed Scopus (283) Google Scholar).Assays Dependent on Heteropolymeric mRNAs (MF-mRNA and MVF-mRNA)Direct Binding of Ac-[14C]Phe-tRNA to the P site in the presence of the MF-mRNA (Table 1, Experiment 1)10 pmol of 80 S ribosomes were incubated in aliquots of 25 μl with 100 pmol of MF-mRNA under assay conditions (30 mM HEPES-KOH, pH 7.6 (0°C), 5 mM MgCl2, 50 mM NH4Cl, 2 mM spermidine, 5 mM β-mercaptoethanol, 1 mM dithioerythritol, 9% glycerol, 0.8 units/μl of RNasin) and incubated for 10 min at 30°C. Then 20 pmol of Ac-[14C]Phe-tRNAs (, 1000 dpm/pmol) were added, and the incubation continued for 20 min at 30°C. The nitrocellulose filtration (tRNA binding) and the puromycin reaction were performed as described elsewhere(23Rheinberger H.-J. Geigenmüller U. Wedde M. Nierhaus K. Methods Enzymol. 1988; 164: 658-670Crossref PubMed Scopus (121) Google Scholar). The backgrounds (minus ribosomes) were 125 dpm (binding) and 376 dpm (puromycin reaction) and were subtracted.Tabled 1 Open table in a new tab Translocation Experiment in the Presence of the MF-mRNA (Table 1, Experiments 2 and 3)90 pmol of 80 S ribosomes were incubated in 225 μl with 900 pmol MF-mRNA and 135 pmol [32P]tRNAiMet (, 1100 dpm/pmol) for 10 min at 30°C. 180 pmol of Ac-[14C]Phe-tRNAPhe (, 1000 dpm/pmol) was added (final volume 350 μl) and incubated for 20 min at 30°C. Thereafter aliquots were withdrawn for the assessment of binding (2 × 50 μl) and puromycin reaction (4 × 25 μl for plus/minus puromycin). To the remaining mixture 15 μl were added containing 50 pmol of EF-2 and GTP (final concentration 1 mM), and the incubation continued for 10 min at 30°C before the binding of tRNAs and the puromycin reaction were determined. Background values were subtracted (binding assays: 22 cpm of 32P and 242 cpm of 14C; puromycin reaction: 396 cpm of 14C).Binding of the Ternary Complex [3H]Phe-tRNA•EF-1α•GTP to 80 S Ribosomes (Table 2)For binding to programmed 80 S, 10 pmol of 80 S ribosomes were incubated in 25-μl aliquots under assay conditions (see above) with 100 pmol of MVF-mRNA for 10 min at 30°C. 12 pmol of [3H]Phe-tRNAPhe •EF-1α•GTP (1800 dpm/pmol) were preincubated with ATP (1 mM), GTP (1 mM), and, where indicated, 10 pmol of EF-3 and then added to the ribosome mixture (final volume 50 μl) and incubated for 20 min at 30°C before the binding was assessed (nitrocellulose filtration). The A site incubation of the i-type was performed as before but included 15 pmol of tRNAVal in the first incubation. For A site incubation of the e-type, the first incubation was performed as described above but with 15 pmol of tRNAiMet. 11.5 μl (assay conditions) containing 20 pmol of [14C]Val-tRNAVal•EF-1α•GTP (570 dpm/pmol) was added, and the incubation continued for 15 min at 30°C. After adding 10 pmol of EF-2 • GTP the translocation was performed (10 min at 37°C). The A site was occupied by the addition of 12 pmol of [3H]Phe-tRNAPhe •EF-1α•GTP (1800 dpm/pmol) and, when indicated, 10 pmol of EF-3•ATP. An incubation followed for 20 min at 30°C. The binding of Val-tRNAVal and the puromycin reaction were determined before and after the translocation reaction. 0.38 Val-tRNAs (6% puromycin reactive) were bound per 80 S before translocation, and 0.35 (69% puromycin reactive) were bound after translocation. The kinetics of the A site binding (Fig. 1) were similarly performed but using [32P]tRNAiMet (450 dpm/pmol) in the first incubation. The binding was determined at 30°C before and at the indicated times (10 s, 5 min, 10 min, and 20 min) after addition of the ternary complex [3H]Phe-tRNAPhe •EF-1α•GTP (1800 dpm/pmol). Each aliquot contained 5 pmol of 80 ribosomes. Background values (minus ribosomes) were subtracted: 31 dpm of 32P, 180 dpm of 14C, 1232 dpm of 3H. In a control experiment the post-translocational complex obtained after EF-2•GTP-dependent translocation was made free from translational factors by centrifugation at 65,000 × g for 2.5 h. The ribosomal pellet was resuspended in binding buffer, and [3H]Phe-tRNA binding in the presence of EF-1α•GTP and EF-3•ATP were performed. The binding of Phe-tRNA and the release of tRNAiMet followed very similar kinetics (data not shown).Tabled 1 Open table in a new tab Chasing of E site bound tRNA: Comparison of Two Post-translocational Complexes (Fig. 2)90 pmol of 80 S ribosomes were incubated in 225 μl (assay conditions) with 900 pmol of MF-mRNA and 135 pmol of [32P]tRNAiMet (850 dpm/pmol) for 10 min at 30°C. 180 pmol of Ac-[14C]Phe-tRNAPhe (1000 dpm/pmol) were added, and the incubation continued for 20 min at 30°C. Then the translocation was performed in the presence of 90 pmol of EF-2 and 1 mM GTP (final volume 480 μl) for 10 min at 30°C. The mixture was separated into various aliquots, and the nonlabeled tRNAs were added in a 10 molar ratio to 80 S (tRNAVal, tRNAiMet, or tRNAThr) followed by an incubation for 5 min at 30°C. The binding of tRNA was assessed in duplicate assays before and after chasing. The amount of bound [32P]tRNAiMet before chasing was taken as 100%. The binding of AcPhe-tRNA as well as its puromycin reactivity was not changed at all by the addition of the nonlabeled tRNAs. The same experiment was also performed with the MVF-mRNA but using [32P]tRNAVal (510 dpm/pmol) as deacylated tRNA.Figure 2:Kinetics of ternary complex binding to ribosomes in the post-translocational state and the effects of EF-3. , [32P]tRNAiMet (450 dpm/pmol); , [14C]Val-tRNAVal (570 dpm/pmol); , ternary complex [3H]Phe-tRNAPhe. EF-1α•GTP (1800 dpm/pmol); •, [32P]tRNAiMet; ▾, [14C]Val-tRNAVal; ○, [3H]Phe-tRNAPhe. POST, ribosomal complex in the post-translocational state; PRE, ribosomal complex in the pre-translocational state.View Large Image Figure ViewerDownload Hi-res image Download (PPT)RESULTSThe Number of tRNA binding Sites and a Functional Test of the E SiteAn analysis of the tRNA binding capacities of yeast ribosomes in the presence of the homopolymeric mRNA poly(U) revealed that up to 1.7 deacylated tRNAPhe and 0.5 AcPhe-tRNAPhe, respectively, could be bound per 80 S ribosome. Since the fraction of ribosomes active in tRNA binding was estimated to be about 60 ± 10%, the conclusion was that yeast ribosomes contain three tRNA binding sites(24Triana F. Nierhaus K.H. Chakraburtty K. Biochem. Mol. Biol. Int. 1994; 33: 909-915PubMed Google Scholar).A rigorous test of this assumption concerning the active fraction of ribosomes can be made with a heteropolymeric mRNA displaying different codons at the various ribosomal sites. Therefore, we designed an mRNA of 46 nucleotides. An mRNA sequence of about this length is protected against RNase attack by the ribosomes(25Legon S. J. Mol. Biol. 1976; 106: 37-53Crossref PubMed Scopus (47) Google Scholar, 26Beyer D. Skripkin E. Wadzack J. Nierhaus K.H. J. Biol. Chem. 1994; 269: 30713-30717Abstract Full Text PDF PubMed Google Scholar). The mRNA contains in the middle two unique codons, AUG and UUC, for methionine (M) and phenylalanine (F), respectively. The sequence of the MF-mRNA is G3(A4G)3A3-AUG-UUC-(A4G)3A3U, which cannot form stable secondary structures (important for efficient tRNA binding) and can be obtained in large amounts by run-off T7 transcription. In the presence of the MF-mRNA, 0.36 AcPhe-tRNAs could be bound per 80 S ribosome (Table 1, Experiment 1), about 20% less binding than obtained in the presence of poly(U) message (0.50 when AcPhe-tRNA was added in saturating amounts; (24Triana F. Nierhaus K.H. Chakraburtty K. Biochem. Mol. Biol. Int. 1994; 33: 909-915PubMed Google Scholar)). This decreased efficiency is typical for direct binding of N-blocked aminoacyl-tRNA to the P site when the binding depends on a codon not located at the 5′-end of the message(18Gnirke A. Geigenmüller U. Rheinberger H.-J. Nierhaus K.H. J. Biol. Chem. 1989; 264: 7291-7301Abstract Full Text PDF PubMed Google Scholar). Of the total amount of bound AcPhe-tRNA, 81% could react with puromycin and thus can be considered to reside at the P site. However, when a deacylated tRNAiMet was prebound, 0.5 AcPhe-tRNAs were found statistically bound per ribosome, and this binding was almost exclusively at the ribosomal A site (90%; Table 1). Obviously, the tRNAiMet fixes the mRNA with the AUG codon at the P site, and as a result, all of the bound mRNA displayed the UUC codon at the A site. The binding value of AcPhe-tRNA at the A site (0.5 per 80 S) found under saturating conditions of the ligand was identical to the maximal binding values observed in the poly(U)-dependent titration experiment (24Triana F. Nierhaus K.H. Chakraburtty K. Biochem. Mol. Biol. Int. 1994; 33: 909-915PubMed Google Scholar). This finding verifies the assumption of the active fraction of 60 ± 10%, which confirms the conclusion that yeast ribosomes have three tRNA binding sites. There is no evidence for a fourth tRNA binding site on yeast ribosomes as claimed for rabbit liver ribosomes(27Rodnina M.V. Wintermeyer W. J. Mol. Biol. 1992; 228: 450-459Crossref PubMed Scopus (18) Google Scholar).AcPhe-tRNA appears in the P site without prebound tRNAiMet but appears almost exclusively at the A site when tRNAiMet is prebound (Table 1, Experiments 1 and 2, respectively). It follows that all ribosomes with AcPhe-tRNA at the A site must have deacylated tRNAiMet at the adjacent P site. After translocation, the fraction of bound tRNAiMet and AcPhe-tRNA molecules did not change significantly, but now the AcPhe-tRNA predominantly resides at the P site (77%) according to the puromycin reaction. These findings also mean that all ribosomes with an AcPhe-tRNA present at the P site carry tRNAiMet at the E site. The tRNA must be firmly bound at the E site, since no tRNA was lost from the ribosome during the manipulations and incubations necessary to carry out the translocation reaction (Table 1, line 3). These results functionally confirm the existence of an E site on yeast ribosomes.An experiment corresponding to that of Table 1but in the presence of EF-3 and ATP yielded the same data, and kinetics of the translocation reaction were identical in the presence or absence of EF-3 (data not shown). We conclude from this experiment that EF-3 does not influence nonenzymatic binding of tRNA to either the P or the A site. Similarly, the peptidyl transferase reaction as well as the EF-2•GTP-dependent translocation reactions were also not influenced by EF-3.Effects of EF-3 on A Site OccupationA reaction of the elongation cycle not yet tested is the enzymatic A site occupation. For this analysis, we chemically synthesized an mRNA 42 nucleotides in length carrying three distinct codons for Met-Val-Phe (MVF-mRNA) in order to allow for E (tRNAiMet) and P site occupation (Val-tRNA) before a Phe-tRNA was added enzymatically. As in the case of MF-mRNA, the sequence of the MVF-mRNA (C17-AUG-GUC-UUC-C16) cannot form a stable secondary structure. The binding of a ternary complex ([3H]Phe-tRNA•EF-1α•GTP) was studied to ribosomes in three different functional states (Table 2). The binding to programmed 80 S ribosomes was not affected by EF-3 (0.34 versus 0.35 Phe-tRNAs bound per ribosome, respectively). However, this binding reaction does not reflect a situation that actually occurs during the elongation cycle. Next, the binding was tested to ribosomes with a deacylated tRNA at the P site and a free E site. This ribosomal state mimics the condition where the initiation phase is completed and the ribosomes are ready to enter the elongation phase, i.e. carrying the initiator Met-tRNAiMet at the P site with a free adjacent E site. However, we have preferred to use deacylated tRNAiMet for P site occupation instead of Met-tRNAiMet in order to focus on A site binding in the next step and to prevent a subsequent formation of a peptide bond. Due to the similarity of the ribosomal state with that of the initiation complex, we termed the corresponding A site occupation the A site binding of the i-type (i for initiation; see (28Hausner T.-P. Geigenmüller U. Nierhaus K.H. J. Biol. Chem. 1988; 263: 13103-13111Abstract Full Text PDF PubMed Google Scholar)). As shown in Table 2, only a slight stimulation is found in the presence of EF-3 (0.40 versus 0.45 Phe-tRNAs bound per 80 S). Therefore, we conclude that A site binding to ribosomes with a free E site is not influenced by EF-3.The ribosomal state tested in the next experiment reflects the standard situation of an A site binding during an elongation cycle, i.e. both P and E sites are occupied with tRNAs (post-translocational state). This state was achieved by an EF-2-dependent translocation of [14C]Val-tRNAVal from the A site to the P site and tRNAiMet from the P site to the E site. Before translocation, only 6% of the bound Val-tRNA reacted with puromycin. However, after translocation, 69% of Val-tRNA became puromycin-reactive, indicating that indeed the majority of ribosomes carrying Val-tRNA were in the post-translocational state. The corresponding A site occupation with [3H]Phe-tRNA was termed A site binding of the e-type (e for elongation; see (28Hausner T.-P. Geigenmüller U. Nierhaus K.H. J. Biol. Chem. 1988; 263: 13103-13111Abstract Full Text PDF PubMed Google Scholar)). EF-3 dramatically affected the enzymatic A site binding of the e-type. In the absence of EF-3, the ternary complex containing Phe-tRNA could hardly occupy the A site, whereas in the presence of EF-3 a 5-fold stimulation of the A site occupation occurs reaching normal binding values (0.08 and 0.38 Phe-tRNAs per 80 S, respectively, see Table 2). The strong EF-3 effects seen with the A site occupation of the e-type is largely dependent on the presence of EF-1α within the ternary complex, since AcPhe-tRNA hardly bound to the A site when the E site was occupied (data not shown).Reciprocal Linkage between A and E SitesThe fact that a deacylated tRNA at the E site prevents the binding of a ternary complex to the A site (in the absence of EF-3, see Table 2) suggests an allosteric linkage between the E and the A sites in the sense of a negative cooperativity. If the observed reciprocal linkage also works in the opposite direction, i.e. occupation of the A site induces a lowering of the E site affinity, the binding of a ternary complex to the A site should trigger the release of deacylated tRNA from the E site. To test this hypothesis, a post-translocational complex was constructed with [14C]Val-tRNA at the P site similar to that described in Table 2but now with 32P-labeled deacylated tRNAiMet at the E site (Fig. 2A). After the addition of [3H]Phe-tRNA together with EF-1α and GTP, very little binding of the ternary complex was observed, with a concomitantly low release of deacylated tRNA from the E site (Fig. 2B). In contrast, in the presence of EF-3 and ATP significant binding of the ternary complex was observed, which was accompanied by a corresponding release of deacylated tRNA from the E site (Fig. 2C). The extent of A site binding and E site release, respectively, was equivalent to the amount of Val-tRNA present at the P site. The rates of binding and release were practically identical, indicating that for each Phe-tRNA bound to the A site, one tRNAiMet was released from the E site. In spite of the strong effects of EF-3 at the A and E sites, the Val-tRNA remained stably bound at the intervening P site, underlining the specificity of the allosteric linkage between the A and E sites. It follows that the reciprocal linkage between A and E sites is bidirectional in the sense of negative cooperativity, i.e. occupation of one site induces a low affinity at the other site as has been reported for prokaryotic ribosomes from E. coli(18Gnirke A. Geigenmüller U. Rheinberger H.-J. Nierhaus K.H. J. Biol. Chem. 1989; 264: 7291-7301Abstract Full Text PDF PubMed Google Scholar, 29Rheinberger H.-J. Nierhaus K.H. J. Biol. Chem. 1986; 261: 9133-9139Abstract Full Text PDF PubMed Google Scholar).EF-3 Is an E Site FactorThe experiments shown in Table 2and Fig. 2demonstrated that significant EF-3 effects were only seen when the E site was occupied (A site occupation of the e-type). Therefore, we asked whether EF-3 could affect the E site directly without a concomitant EF-1α-dependent A site binding. To this end, two post-translocational complexes were prepared using either the MF-mRNA (Fig. 3A) or the MVF-mRNA (Fig. 3B). In both cases, Ac-[14C]Phe-tRNA was present at the P site, carrying either a [32P]tRNAiMet (Fig. 3A) or a [32P]tRNAVal (Fig. 3B) at the adjacent E sites. The point of this experimental design is as follows: a deacylated tRNA, which is cognate to the E site codon in one system, is "near cognate" in the other. For example, tRNAMet is cognate to the E site codon in the MF-mRNA system and near cognate to that of the MVF-mRNA system; the opposite is true for tRNAVal (note that the central base of a codon is the most critical for the recognition process and that the first base of both codons under observation is a purine; see (30Osawa S. Jukes T.H. Watanabe K. Muto A. Microbiol. Rev. 1992; 56: 229-264Crossref PubMed Google Scholar)).Figure 3:Chasing of E site bound tRNA: comparison of two post-translocational complexes containing [32P]tRNAiMet or [32P]tRNAVal. , [32P]tRNAiMet (850 dpm/pmol); , Ac-[14C]Phe-tRNAPhe (1000 dpm/pmol); , [32P]tRNAVal (510 dpm/pmol). The chasing exclusively affected the deacylated tRNA in the E site; the AcPhe-tRNA in the P site was not affected. Chasing agents (10 × over ribosomes) are as follows: tRNAVal (solid bar), [32P]tRNAiMet (openbar), and tRNAThr (shadedbar). For further explanations see "Experimental Procedures."View Large Image Figure ViewerDownload Hi-res image Download (PPT)The "tightness" of the E site binding and the direct effect of EF-3 on this site were tested in the following way. We assessed chasing effects at the E site by adding nonlabeled tRNAiMet or tRNAVal in a 10 molar excess over the ribosomes. The data presented in Fig. 3demonstrate that under all conditions the chasing substrate cognate to the E site codon clearly exceeded the effects seen with the near cognate tRNA. Noncognate tRNAThr (codon: ACC) had no effect in the absence of EF-3. The striking dependence on the anticodon character of the chasing substrate identifies codon-anticodon interaction as an important determinant of the binding of deacylated tRNA to the E site. A second important feature of the experiments shown in Fig. 3, A and B, is the strong effect induced by EF-3•ATP. The factor facilitates the chasing by both cognate and near cognate tRNAs, and now even the noncognate tRNA has a low but significant effect. The EF-3 effect is pronounced and E site-specific, i.e the EF-3 effect is observed in the absence of aminoacyl-tRNA and EF-1α.The results shown in Fig. 4demonstrate that the E site-specific effect of EF-3 requires hydrolysis of ATP. There is no EF-3-dependent release of deacylated tRNA from the E site in the absence of ATP or in the presence of AMPPNP, the noncleavable analogue of ATP.Figure 4:Chasing of the E site bound tRNA: dependence on EF-3 and ATP. The post-translocational state was made as in Fig. 3B but with limiting amounts of 14 μM GTP. For chasing, a 10 molar ratio of nonlabeled tRNAVal was added as well as EF-3 (1 pmol per 80 S) and, where indicated, ATP (1 mM) or AMP-PNP (1.5 mM). After 5 min at 30°C the binding was determined by a nitrocellulose filter-binding assay.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONThe experimental data reveal that the three essential features of the allosteric three-site model for the elongation cycle (31Nierhaus K.H. Biochemistry. 1990; 29: 4997-5008Crossref PubMed Scopus (114) Google Scholar) are also valid for the yeast ribosomes. 1) In addition to the A and P sites, a third site (E site) exists for the yeast ribosomes. 2) The E site is specific for deacylated tRNA and is reciprocally linked to the ribosomal A site, i.e. occupation of the A site reduces the affinity for tRNA of the E site and vice versa. 3) The deacylated tRNA at the E site undergoes codon-anticodon interaction. The allosteric three-site model was first derived from experimental data obtained with ribosomes of the eubacteria E. coli(18Gnirke A. Geigenmüller U. Rheinberger H.-J. Nierhaus K.H. J. Biol. Chem. 1989; 264: 7291-7301Abstract Full Text PDF PubMed Google Scholar) and then demonstrated for archaebacterial ribosomes from Halobacterium halobium(32Saruyama H. Nierhaus K.H. Mol. & Gen. Genet. 1986; 204: 221-228Crossref Scopus (21) Google Scholar), and as shown here, it is also valid for the eukaryotic yeast ribosomes. The model thus probably describes universal features of elongating ribosomes.The possible effect of EF-3 on ribosomal function was tested in different assays covering the complete elongation cycle. The factor affected neither the translocation extent and speed nor tRNA binding to programmed ribosomes containing no tRNA or one tRNA bound to the P site. In contrast, the EF-1α-dependent binding of aminoacyl-tRNA to a ribosomal complex containing two tRNAs bound to the P and E sites required the presence of EF-3 and ATP (Table 2 and Fig. 2). Additionally, a direct involvement of EF-3 with the ribosomal E site function was demonstrated with the factor-dependent increase of chasing a tRNA from this site (Fig. 3). The EF-3-induced hydrolysis of ATP was essential for this effect (Fig. 4), which constitutes the first reported activity of this factor in the absence of EF-1α.The function of EF-3 can be neatly explained in the frame of the allosteric three-site model. EF-3 is essential for the A site occupation of the e-type. If the reciprocal linkage between the A and E sites is a stringent feature of the elongation cycle, a release of deacylated tRNA from the E site is a prerequisite for A site binding. The observed EF-3 effects can thus be reconciled as follows. Deacylated tRNA is tightly bound to the E site; EF-3-dependent ATP hydrolysis is required to enable the release of tRNA from this site by binding the cognate ternary complex to the A site. The binding energy contributed by EF-1α is obviously essential for this allosteric transition from the post- to the pretranslocational state, since acylated tRNA cognate to the A site codon cannot trigger this reaction in the absence of EF-1α or in the presence of EF-3 alone.In higher eukaryotes a ribosomal ATPase/GTPase has been described(33Grummt F. Grummt I. Erdmann V. Eur. J. Biochem. 1974; 43: 343-348Crossref PubMed Scopus (38) Google Scholar). It is tempting to speculate that EF-3 has become an integral component of the ribosome and exerts its function as a ribosomal protein. In prokaryotic ribosomes the recently described second EF-Tu-dependent GTP hydrolysis required for the binding of cognate aminoacyl-tRNA to the A site (34Ehrenberg M. Rojas A.M. Weiser J. Kurland C.G. J. Mol. Biol. 1990; 211: 739-749Crossref PubMed Scopus (73) Google Scholar, 35Bilgin N. Claesens F. Pahverk H. Ehrenberg M. J. Mol. Biol. 1992; 224: 1011-1027Crossref PubMed Scopus (107) Google Scholar, 36Weijland A. Parmeggiani A. Science. 1993; 259: 1311-1314Crossref PubMed Scopus (97) Google Scholar) could fulfill a function analogous to that of the EF-3-dependent ATP hydrolysis. It is thus possible that the EF-3 function defined here for yeast ribosomes reflects a universal feature of protein biosynthesis INTRODUCTIONRibosomes from organisms of all kingdoms require two factors in the course of the reactions of the elongation cycle; elongation factor 1α (EF-1α) 1The abbreviations used are: EFelongation factorAMP-PNPadenosine 5′-(β,γ-imino)triphosphate. is responsible for the codon-dependent selection of the cognate aminoacyl-tRNA at the ribosomal A site and elongation factor 2 (EF-2) for the translocation reaction. The discovery of a third elongation factor (EF-3) essential for the elongation cycle in the yeast Saccharomyces cerevisiae was therefore a surprise(1Skogerson L. Wakatama E. Proc. Nat. Acad. Sci. U. S. A. 1976; 37: 73-76Crossref Scopus (60) Google Scholar). Furthermore, this factor is a ribosome-dependent ATPase, which also accepts GTP and ITP as substrates and to a lesser extent pyrimidine nucleotides(2Dasmahapatra B. Chakraburtty K. J. Biol. Chem. 1981; 256: 9999-10004Abstract Full Text PDF PubMed Google Scholar, 3Uritani M. Miyazaki M. J. Biochem. (Tokyo). 1988; 103: 522-530Crossref PubMed Scopus (42) Google Scholar, 4Kamath A. Chakraburtty K. J. Biol. Chem. 1989; 264: 15423-15428Abstract Full Text PDF PubMed Google Scholar). This wide substrate specificity of EF-3 is one principal difference from other translational factors, which show a stringent requirement for GTP(5Moldave K. Annu. Rev. Biochem. 1985; 54: 1109-1149Crossref PubMed Scopus (357) Google Scholar).EF-3 from S. cerevisiae consists of a single polypeptide chain of 1044 amino acids(6Chakraburtty K. Fernandes P. New Approaches to Anti-fungal Agents. Birkhäuser Verlag, Basel1992: 114-142Google Scholar, 7Sandbaken M. Lupisella J.A. DiDomenico B. Chakraburtty K. J. Biol. Chem. 1990; 265: 15838-15844Abstract Full Text PDF PubMed Google Scholar). The genes encoding the EF-3 analogues in Candida albicans and in Pneumocystis carinii, higher fungi than yeast, have also recently been isolated and sequenced (8Qin S. Xie A. Banato C.M. McLaughlin C. J. Biol. Chem. 1990; 265: 1903-1912Abstract Full Text PDF PubMed Google Scholar, 9DiDomenico B.J. Lupisella J. Sandbaken M. Chakraburtty K. Yeast. 1992; 8: 337-352Crossref PubMed Scopus (36) Google Scholar, 10Ypma-Wong M. Fonzi W. Sypherd P.S. Infect. Immun. 1992; 60: 4140-4145Crossref PubMed Google Scholar). The deduced amino acid sequences from these two species show over 75% identity with the EF-3 from S. cerevisiae. The Candida EF-3 gene could supplement the disrupted EF-3 gene in S. cerevisiae, demonstrating the functional identity of EF-3 among different fungi(9DiDomenico B.J. Lupisella J. Sandbaken M. Chakraburtty K. Yeast. 1992; 8: 337-352Crossref PubMed Scopus (36) Google Scholar). The activity of EF-3 is distinct from that of EF-1α and EF-2, since EF-1α and EF-2 from yeast can functionally replace their counterparts in the presence of ribosomes from rat liver or wheat germ, whereas an EF-3 dependence is only observed with yeast ribosomes(11Skogerson L. Engelhardt D. J. Biol. Chem. 1977; 252: 1471-1475Abstract Full Text PDF PubMed Google Scholar, 12Chakraburtty K. Kamath A. Int. J. Biochem. 1988; 20: 581-590Crossref PubMed Scopus (29) Google Scholar). This dependence follows the source of 40 S subunits as suggested by experiments with hybrid ribosomes(12Chakraburtty K. Kamath A. Int. J. Biochem. 1988; 20: 581-590Crossref PubMed Scopus (29) Google Scholar). A monoclonal antibody against EF-3 blocks in vitro translation of poly(U) as well as natural mRNA, and evidence was obtained that EF-3 was associated with polysomes rather than with ribosomal subunits and that the protein is required for every cycle of the elongation of the polypeptide chain(13Hutchinson J.S. Feinberg B. Rotwell T.C. Moldave K. Biochemistry. 1984; 23: 3055-3063Crossref PubMed Scopus (25) Google Scholar). Polyclonal antibodies did not cross-react with extracts from mammals (liver and reticulocytes) or plants (wheat germ) and, accordingly, did not affect the respective translational systems(12Chakraburtty K. Kamath A. Int. J. Biochem. 1988; 20: 581-590Crossref PubMed Scopus (29) Google Scholar).The actual step of the elongation cycle promoted by EF-3 has not been identified. The factor is not involved in the nucleotide exchange reactions of either EF-1α or EF-2(4Kamath A. Chakraburtty K. J. Biol. Chem. 1989; 264: 15423-15428Abstract Full Text PDF PubMed Google Scholar). Slight stimulations of some other partial reactions of the elongation cycle including peptide bond formation and translocation have been reported(14Miyazaki M. Uritani M. Kagiyama H. J. Biochem. (Tokio). 1988; 104: 445-450Crossref PubMed Scopus (17) Google Scholar, 15Miyazaki M. Kagiyama H. J. Biochem (Tokyo). 1990; 108: 1001-1008Crossref PubMed Scopus (17) Google Scholar). More detailed examinations revealed that aminoacyl-tRNA binding to the A site was stimulated by EF-3 but only in the presence of catalytic amounts of EF-1α(4Kamath A. Chakraburtty K. J. Biol. Chem. 1989; 264: 15423-15428Abstract Full Text PDF PubMed Google Scholar), which does not easily explain the absolute requirement found for EF-3 in vivo(7Sandbaken M. Lupisella J.A. DiDomenico B. Chakraburtty K. J. Biol. Chem. 1990; 265: 15838-15844Abstract Full Text PDF PubMed Google Scholar).
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