Functional Importance of the 3 (cid:1) -Terminal Adenosine of tRNA in Ribosomal Translation*

The universally conserved 3 (cid:1) -terminal CCA sequence of tRNA interacts with large ribosomal subunit RNA during translation. The functional importance of the interaction between the 3 (cid:1) -terminal nucleotide of tRNA and the ribosome was studied in vitro using mutant in vitro transcribed tRNA Val A76G. Val-tRNA CCG does not support polypeptide synthesis on poly(GUA) as a mes-sage. However, in a co-translation system, where Val-tRNA CCG represented only a small fraction of total Val- tRNA, the mutant tRNA is able to transfer valine into a polypeptide chain, albeit at a reduced level. The A76G mutation does not affect binding of Val- or NAcVal-tRNA CCG to the A- or P-sites as shown by efficient pep- tide bond formation, although the donor activity of the mutant NAcVal-tRNA CCG in the peptidyl transfer reac- tion is slightly reduced compared with wild-type NAcVal-tRNA. Translocation of 3 (cid:1) -CCG-tRNA from the P-to the E-site is not significantly influenced. However, the A76G mutation drastically inhibits translocation of peptidyl-tRNA G 76 from the ribosomal A-site to the P-site, which apparently explains its failure to support cell-free protein synthesis. Our results indicate that the identity of the mycin reaction. Mixes were incubated in the presence of 1 m M puromy- cin at 0 °C for 30 min. The reaction was stopped by the addition of sodium hydroxide (0.6 M final concentration) and incubated 15 min at 37 °C to hydrolyze any remaining aminoacyl-tRNA. After neutraliza-tion with 0.2 ml of 1 M potassium phosphate (pH 7.6) NAc[ 3 H]Val- puromycin was extracted with 1 ml of ethyl acetate. The amount of NAc[ 3 H]Val-puromycin formed was determined by scintillation count- ing of the ethyl acetate phase.

The 3Ј-CCA sequence of tRNA is a universal ligand for protein biosynthesis; it is recognized by aminoacyl-tRNA synthetases, EF-Tu, and 23 S rRNA (1,2). Experiments using in vitro transcribed tRNA Val variants demonstrated the importance of the 3Ј-CCA sequence for aminoacylation (3,4) and its significance in formation of the ternary complex between Val-tRNA, EF-Tu, and GTP (5). On ribosomes, the CCA end of tRNA interacts with 23 S rRNA at all ribosomal tRNA binding sites (6). The importance of the CCA end in ribosome-catalyzed peptide bond formation is well established (1,7). Chemically synthesized aminoacyl oligonucleotides were used to demonstrate the significance of the 3Ј-CCA sequence as a peptide acceptor during peptide bond formation on ribosomes (8). E. coli tRNA Val with mutations in the 3Ј-CCA sequence inhibits the peptidyltransferase activity of the ribosome (9). These findings were rationalized by showing functional base pairing of C 74 with G 2252 of 23 S rRNA at the donor site of the ribosomal peptidyltransferase center (10) and of C 75 with G 2553 at the acceptor site (11)(12)(13).
Mutation of C 74 to U 74 in tRNA His derepresses the histidine biosynthetic operon of Salmonella typhimurium (14). Mutants of Escherichia coli tRNA 1 Val with 3Ј-GCA or 3Ј-ACA promote Ϫ1 frameshifting and suppress a wide variety of nonsense mutants (14). It has also been proposed that substitution of the A 76 of tRNA affects binding of deacylated tRNA to the ribosomal E-site (15). These findings show the importance of the 3Ј-CCA end of tRNA in maintaining the reading frame during translation and suggest that the 3Ј-CCA end is involved in ribosomal translocation.
In this paper, we analyze the functional importance of the 3Ј-terminal A of tRNA for ribosomal translation. The results show that substitution of the 3Ј A of tRNA Val with G blocks translocation of peptidyl-tRNA from the ribosomal A-site to the P-site and inhibits the peptidyl transfer reaction at the ribosomal donor (P) site.

Preparation of tRNAs, Poly(GUA), and Enzymes
Transfer RNA Val (anticodon UAC) was synthesized in vitro by T7 RNA polymerase-catalyzed run-off transcription from the phagemid pFVAL119 linearized by FokI (A76) or from pFVALG76 linearized by MspI (16). This method was shown to produce the expected nucleotide at the 3Ј-terminus (17). tRNA Val variants were purified by size exclusion chromatography on Sephadex G-50 and gel electrophoresis in 7% urea-PAGE. Total E. coli tRNA was from Roche Molecular Biochemicals. tRNAs are designated as follows: tRNA CCA (in vitro transcribed wild-type tRNA Val ), tRNA CCG (in vitro transcribed tRNA Val with the mutation A76G), tRNA bulk (total (unfractionated) E. coli tRNA), and tRNA Val (purified isoacceptors of valine-specific E. coli tRNA). 1 g of tRNA Val was taken as equal to 40 pmol.
DNA encoding the sequence poly(GTA) 44 was the kind gift of Dr. T. Tenson (University of Tartu). The poly(GTA) 44 was amplified by PCR and cloned under the control of the T7 late promoter in pLITMUS38 between the ApaI and EcoRV sites. The plasmid was cleaved by BspTI, and poly(GUA) 44 was prepared by in vitro transcription with T7 RNA polymerase. Poly(GUA) mRNA has the sequence 5Ј-GGCCCGUA-GA(GUA) 44 . Transcripts were purified by gel filtration chromatography on Sephadex G-50. 1 g of poly(GUA) 44 was taken as equal to 25 pmol.

Aminoacylation
Transfer RNAs were aminoacylated by T. aquaticus ValRS as described by Liu and Horowitz (3), using [ 3 H]Val or [ 14 C]Val (Amersham Biosciences) with specific activities of 30,000 and 500 dpm/pmol, respectively. Charging levels were 20 -25 pmol of Val/g of tRNA for in vitro transcribed tRNA variants and 1.5 pmol of Val/g of total E. coli tRNA. N-Acetylation of Val-tRNA variants was according to Haenni and Chapeville (21).

In Vitro Poly(GUA)-directed Translation
Ribosomes were isolated according to Rodnina and Wintermeyer (22). In vitro translation assays were carried out in buffer A (20 mM Tris⅐HCl, pH 7.6, 160 mM NH 4 Cl, 12 mM MgCl 2 , 5 mM 2-mercaptoethanol), essentially as described by Gavrilova et al. (23). Translation initiation complexes (mix R) were prepared by mixing 25 pmol of poly(GUA) 44 , as mRNA, with 10 pmol of 70 S ribosomes in 15 l of buffer A and incubating at 37°C for 10 min. Mix T, which contained (in 20 l of buffer A) 0 -40 pmol of Val-tRNA variant, 10 g of EF-Tu, 2 g of EF-G, and 2.5 mM GTP, was incubated for 5 min at 30°C. In co-translation experiments, mix R contained 0 -8 pmol of ribosomes, 1 pmol of [ 3 H]Val-tRNA variant, and 25 pmol of poly(GUA); mix T contained 20 pmol of E. coli tRNA bulk , charged with [ 14 C]Val, elongation factors, and GTP as above. If NAcVal-tRNA variants were used to initiate translation, mix R contained 20 pmol of NAc In all translation experiments, reactions were started by combining mix R with mix T. After a 20-min incubation at 37°C, the reactions were stopped by the addition of 1.5 ml of 5% trichloroacetic acid, and the samples were heated at 95°C for 20 min. Precipitates were collected on glass fiber filters, which were dried and counted in a scintillation spectrometer.

Dipeptide Synthesis Assay
Acceptor and donor activities of Val-tRNA in ribosomal peptide bond formation were analyzed by measuring dipeptide synthesis (in the absence of EF-G) using in vitro transcribed Val-tRNA and NAcVal-tRNA variants. Two mixes were prepared. Mix R, containing (per 30 l) 25 pmol of poly(GUA) 44 , 5 pmol of 70 S ribosomes, and 10 pmol of NAc[ 3 H]Val-tRNA in buffer A, was preincubated for 10 min at 37°C. Mix T, containing (per 20 l) 0 -8 pmol of [ 14 C]Val-tRNA, 220 pmol of EF-Tu, 2.5 mM GTP, 2.5 mM PEP, and 10 units of PEP kinase in buffer A, was preincubated 5 min at 30°C. Dipeptide bond formation was initiated by combining mix R and mix T. After incubating for 10 min at 37°C, the reaction was stopped by the addition of NaOH to a final concentration of 0.6 M. Samples were further incubated for 20 min at 37°C to hydrolyze aminoacyl-and peptidyl-tRNA and 200 l of 5 N H 2 SO4 were then added to lower the pH to Ͻ1. Translocation EF-G-dependent translocation was analyzed according to Watanabe (24).
A-to P-site Translocation-To assay the effect of G 76 on the translocation of tRNA from the ribosomal A-site to the P-site, the ribosomal Pand E-sites were filled with deacylated tRNA CCA in the presence of P-to E-site Translocation-This assay is similar to that of the A-to P-site translocation assay except that the ribosomal P-and E-sites were occupied with either deacylated tRNA CCA or deacylated tRNA CCG in the presence of poly(GUA) by preincubating 25 l of mix R (5 pmol of 70 S ribosomes, 25 pmol of poly(GUA) 44 , 20 pmol of tRNA CCA or tRNA CCG in buffer A) at 37°C for 10 min. Subsequently, 10 pmol of NAc[ 3 H]Val-tRNA CCA were added and allowed to bind to the ribosomal A-site at 37°C for 20 min. Translocation was initiated as described for the A-to P-site translocation assay, and the amount of NAc[ 3 H]Val-tRNA CCA translocated to the P-site was measured by the puromycin reaction. The A-site tRNA can only go to the P-site after P to E translocation has occurred; thus, if tRNA CCG blocks this movement, then A to P translocation cannot occur.

Translation of Poly(GUA) by in Vitro Transcribed
Val-tRNA Val Variants-Prior work showed that in vitro transcribed tRNA Val variants C 76 and U 76 , while readily accepting valine, are inactive in polypeptide synthesis (3,9) largely because of a decreased affinity for EF-Tu⅐GTP (5). In contrast, tRNA Val with a 3Ј-terminal guanine (G 76 ), which is a poor substrate for valyl-tRNA synthetase but can be aminoacylated at high enzyme concentrations, does function in translation as shown by its ability to transfer valine into poly(Val,Phe) in a poly(U 3 ,G)-directed reaction, but only if Phe-tRNA is also present (3). We have therefore restricted our studies to the G 76 variant of tRNA Val . The ability of this mutant tRNA to support synthesis of poly(Val) on E. coli 70 S ribosomes in an in vitro poly(GUA)directed system was compared with that of wild-type tRNA Val (A 76 ). Aminoacylated tRNAs were utilized to eliminate effects due to differences in the rates of aminoacylation (3). Purified E. coli translation elongation factors EF-Tu and EF-G were used in these experiments, and no aminoacyl-tRNA synthetases or CCA-adding enzyme activity were present. Therefore, incorporation of valine into polypeptides is exclusively from added Val-tRNA.
Valine is incorporated into polypeptide from both native (modified) E. coli [ 14 C]Val-tRNA bulk and from in vitro transcribed [ 14 C]Val-tRNA CCA at levels proportional to the [ 14 C]Val-tRNA added (Fig. 1). In control experiments without poly(GUA), no trichloroacetic acid-precipitable valine incorporation was observed (results not shown). Under conditions of the experiment the unmodified tRNA Val transcript is quite active in poly(Val), indicating that modified nucleotides are not essential for translation on the ribosome. Others have observed that tRNAs lacking modified nucleotides are only marginally less efficient in translation than native (modified) tRNAs (26 -29) (for a review, see Ref. 30).
In contrast to wild-type Val-tRNA Val , the mutant [ 14 C]Val-tRNA CCG is completely inactive in poly(GUA)-directed translation (Fig. 1). Although the G 76 variant of tRNA Val alone is not active, it does transfer valine into polypeptides in a co-trans-  (Fig. 2A). This result is consistent with the finding of Liu and Horowitz (3) that the Val-tRNA CCG transcript can participate in ribosomal translation in vitro. Comparison with incorporation of [ 3 H]Val from wild-type [ 3 H]Val-tRNA CCA into polypeptides in the co-translation system demonstrates that the G 76 mutant is considerably less active (Fig. 2, compare A and B). Furthermore, incorporation of [ 14 C]Val (from wild-type [ 14 C]Val-tRNA bulk ) into peptides is one-third lower in the presence of the G 76 tRNA Val mutant (Fig.  2, A and B), indicating that Val-tRNA CCG inhibits poly(GUA) translation by native (A 76 ) Val-tRNA Val .
The possibility that valine is transesterified from tRNA CCG to tRNA CCA was examined by adding deacylated wild-type tRNA CCA to a reaction in which [ 14 C]Val-tRNA CCG is the only source of valine. If transesterification is responsible for incorporation of radioactivity from Val-tRNA CCG into poly(Val) in the co-translation assay, incorporation should be stimulated by the added deacylated tRNA CCA . Such stimulation was not observed (data not shown), and we conclude that transesterification of valine from mutant to wild-type tRNA does not occur in our system.
To test the donor activity of Val-tRNA variants in poly(GUA)-directed translation, NAcVal-tRNA was added to the reaction. N-acetyl aminoacyl-tRNA can serve only as a donor substrate for peptide bond formation and acts as an initiator tRNA in this assay. For these experiments, ribosomal initiation complexes were formed by incubating NAc[ 3 H]Val-tRNA CCA or NAc[ 3 H]Val-tRNA CCG with ribosomes and poly(GUA), followed by a second incubation step in the presence of either [ 14 C]Val-tRNA CCA or [ 14 C]Val-tRNA CCG , elongation factors, and GTP. It is evident, from the results, shown in Fig. 3 (Fig. 3, A and B). In contrast, polypeptide synthesis does not occur when [ 14 C]Val-tRNA CCG serves as the elongator tRNA, independent of which tRNA Val variant is used for initiation (Fig. 3, C and D). These results show that the G 76 mutant of tRNA Val is active as a donor substrate for ribosomal peptide bond formation. It is not clear, however, whether the G 76 tRNA Val mutant can serve as an acceptor at the peptidyltransferase center, because reactions other than peptide bond formation may be affected by the mutation. The results suggest that the G 76 tRNA Val mutant cannot participate in poly(GUA)directed translation due to a defect in peptide chain elongation.
Dipeptide Synthesis-The inability of the G 76 tRNA Val mutant to function in poly(GUA)-directed translation was examined further by analyzing the effects of this mutation on peptidyltransferase activity as measured by dipeptide formation.
To investigate the acceptor activity of Val-tRNA variants in peptide bond synthesis, NAc[ 3 H]Val-tRNA CCA (A76) was bound to the ribosomal P-site in a codon-dependent manner, and dipeptide synthesis was followed after the addition of increasing amounts of either [ 14 C]Val-tRNA CCA or [ 14 C]Val-tRNA CCG, complexed with EF-Tu and GTP. Fig. 4A shows that wild-type tRNA Val and the G 76 variant are active as acceptors in peptide bond formation. Clearly the A to G transition at position 76 of tRNA Val does not affect acceptor activity of tRNA Val in our system. This result agrees with the observation that substitutions in the CCA sequence have only small effects on the acceptor activity of chemically synthesized aminoacyl-oligonucleotides in the ribosomal peptidyltransferase reaction (8).
Donor activity of the G 76 tRNA Val mutant, was determined by binding either NAc[ 3 H]Val-tRNA CCG or wild-type NAc[ 3 H]Val-tRNA CCA to poly(GUA)-programmed 70 S ribosomes and measuring the extent of dipeptide synthesis after incubation with increasing concentrations of [ 14 C]Val-tRNA CCA -EF-Tu-GTP. The results show that both substrates, NAcVal-tRNA CCG and NAcVal-tRNA CCA are active as donors in peptide bond formation, although the G 76 variant is slightly less efficient (Fig. 4B). This result agrees with the observation that NAc[ 3 H]Val-tRNA CCG can serve as initiator tRNA in poly(GUA)-directed translation (Fig. 3B). NAcVal-tRNA CCG was also shown to be active as the donor in ribosome-catalyzed peptide bond formation when puromycin was the acceptor substrate (9).
The dipeptide synthesis experiments show that the G 76 tRNA Val mutant can participate in ribosomal peptide bond formation both as an acceptor and as a donor substrate. Therefore, the inability of tRNA CCG to function in poly(GUA)-directed translation cannot be attributed to a defect in peptide bond formation per se.

tRNA 3Ј-Terminal Adenosine in Ribosomal Translation
A-to P-site Translocation-To determine the effects of the 3Ј-terminal nucleotide of tRNA Val on the movement of tRNA from the ribosomal A-site to the P-site, the P-and E-sites were preincubated with deacylated wild-type tRNA CCA under conditions where the P-and E-sites are known to bind tRNA ( (Fig. 5B). At 37°C, EF-G-dependent translocation of NAc[ 3 H]Val-tRNA CCG does occur, but at a much lower level than that of wild-type NAc[ 3 H]Val-tRNA CCA (Fig. 5C).
In control experiments, NAc[ 3 H]Val-tRNA CCA or NAc-[ 3 H]Val-tRNA CCG bound directly to the P-site readily react with puromycin to the same degree (data not shown). Thus, the failure of NAc[ 3 H]Val-tRNA CCG to react with puromycin is due to inefficient translocation and not to its inability to react with puromycin from the P-site.
P-to E-site Translocation-The 3Ј-terminal nucleotide of tRNA has been shown to play a role in the binding of deacylated tRNA to the ribosomal exit site (E-site) (15,32). Therefore, substitution of A 76 may inhibit movement of deacylated tRNA from the ribosomal P-site to the E-site during translocation and thereby inhibit translation.
The effect of the G 76 mutation in tRNA Val on the translocation of tRNA from the ribosomal P-site to the E-site was tested in a manner similar to that for A-to P-site translocation. Poly(GUA)-programmed 70 S ribosomes were incubated with either deacylated tRNA CCA or tRNA CCG under conditions where the P-and E-sites are known to bind tRNA (6, 31-34). In a second incubation step, wild-type (A 76 ) NAc[ 3 H]Val-tRNA CCA was bound to the ribosomal A-site (see Fig. 6A). Similar amounts of NAc[ 3 H]Val-tRNA CCA were bound to ribosomes with both prebound tRNA Val variants (10,000 dpm with tRNA CCA and 12,000 dpm with tRNA CCG ), as determined by nitrocellulose binding assay (data not shown). Translocation of NAc[ 3 H]Val-tRNA CCA from the A-site to the puromycin reactive P-site was promoted by the addition of increasing concentrations of EF-G. Elongation factor-dependent translocation of NAc[ 3 H]Val-tRNA CCA from the A-site to the P-site, as measured by the puromycin reaction, is essentially the same, regardless of which tRNA Val variant is present at the P-and E-sites (Fig. 6B), clearly indicating that the G 76 tRNA Val mutant is displaced from the ribosomal P-and E-sites as readily as wild-type (A 76 ) tRNA Val .
Based on these results, we can conclude that replacing A 76 in tRNA Val with G 76 interferes with EF-G-dependent ribosomal A-to P-site translocation, and this explains the failure of Val-tRNA CCG to function in poly(GUA)-directed translation. DISCUSSION In this study, we have examined the functional role of the 3Ј-terminal nucleotide of tRNA Val in ribosomal translation by comparing the activity of in vitro transcribed wild-type tRNA Val (A 76 ) with that of the G 76 mutant in translation of the cognate (GUA) codon and in individual steps of the polypeptide elongation cycle. Our results show that the G 76 Val-tRNA Val mutant is completely inactive in poly(GUA)-directed translation when present alone (Fig. 1), suggesting that the A76G mutation affects a step in polypeptide synthesis on the ribosome. Cotranslation experiments, carried out in the presence of excess native (A 76 ) tRNA Val , demonstrate that the mutant tRNA CCG can function in poly(GUA)-directed translation although with reduced efficiency (Fig. 2A).
To identify the step affected by the G 76 mutation, each phase of the polypeptide elongation cycle was individually investigated. Studies of the activity of tRNA CCG in dipeptide synthesis show that the G 76 mutation does not significantly affect the acceptor activity of Val-tRNA in the peptidyltransferase reaction (Fig. 4A). Substitutions in the CCA sequence have also been shown to have only small effects on the acceptor activity of aminoacyl-oligonucleotides in the peptidyl transfer reaction (8).
In contrast, the donor activity of NAcVal-tRNA CCG is reduced by 20 -30% compared with that of NAcVal-tRNA CCA in both poly(GUA)-directed translation (Fig. 3) and in the dipeptide synthesis assay (Fig. 4B). Evidently, the identity of the 3Ј-nucleotide of tRNA is more important at the donor site and less important at the acceptor site of the ribosomal peptidyltransferase center. Crystallographic data of aminoacyl-and peptidyl-tRNA analogs bound to Haloarcula marismortui 50 S ribosomes (7) indicate that N-1 of A 76 of the tRNA at the donor site acts as a hydrogen bond acceptor from the 2Ј-OH of A 2450 of the 23 S rRNA and the base stacks on the ribose of A 2451 . A guanine at position 76 of tRNA can still stack efficiently on the ribose of A 2451 but will have difficulty forming the appropriate hydrogen bonds with the 2Ј-OH group of A 2450 because its N-1 is protonated, unlike that of A 76 . This may account for the reduced donor activity of the G 76 variant of tRNA Val at the peptidyl transfer center.
It has been proposed that binding of tRNA to the E-site promotes movement of the tRNA-mRNA complex with respect to the ribosome (15). The removal or substitution of A 76 decreases the affinity of the ribosomal E-site for tRNA at least 100-fold (15,32,35). Failure of the G 76 mutant of tRNA Val to translate poly(GUA) may be due to the inability of the G 76 mutant to bind correctly to the E-site, thus blocking movement of tRNA from the P-to the E-site. Our results, however, reveal that the A76G mutation does not interfere with the transloca- tion of tRNA from the P-site to the E-site (Fig. 6B). In this connection, it is interesting to note that the 3Ј-end of deacylated tRNA that is formed after transpeptidation does not immediately progress to the E-site but remains temporarily at the peptidyltransferase center, as shown by recent cross-linking experiments (6). It is possible that the G 76 tRNA Val mutant can dissociate from ribosomal P-site without stably binding to the E-site.
The most striking result of the A 76 to G 76 substitution in tRNA Val is the nearly complete inhibition of A-to P-site tRNA translocation. The experiments presented in Fig. 5 clearly demonstrate that translocation of the peptidyl-tRNA analog, NAc[ 3 H]Val-tRNA, from the A-to the P-site is severely inhibited by the A76G mutation, thus accounting for the inability of the G 76 mutant of tRNA Val to translate poly(GUA). This result is in contrast to the ability of the G 76 mutant of tRNA Val to bind to both the A-and the P-sites, as inferred from its activity both as an acceptor and a donor in peptide bond formation (Fig. 4). According to the hybrid state model, the 3Ј-end of tRNA moves concomitantly with peptide bond formation from the acceptor to the donor site leading to the P/A hybrid state (36). This movement and the related conformational changes (37) are expected to depend on correct interaction between the CCA end of tRNA and 23 S rRNA. The affinity of aminoacyl-oligonucleotides for the ribosomal donor site is higher by 1 order of magnitude compared with the acceptor site (38,39). This affinity gradient can be essential for movement of the CCA end of peptidyl-tRNA during translocation (40). If the G 76 variant of peptidyl-tRNA does not bind correctly to the ribosomal donor site, as implied by its reduced donor activity (Figs. 3 and 4B), it can affect movement of the CCA end of tRNA and thereby inhibit translocation. On the other hand, it is possible that A 76 of peptidyl-tRNA is specifically recognized by 23 S rRNA or a ribosomal protein during the translocation reaction. One candidate for such recognition is nucleotide A 2602 , which is disordered in the empty 50 S subunit and becomes positioned between the CCA bound at the A-site and the CCA bound at the P-site after tRNA binding (7). A second candidate is ribosomal protein L 27 , which can be cross-linked to A 76 at both the A-and the P-sites (41).
Feinberg and Joseph have recently identified two 2Ј-OH groups, at positions 71 and 76, which are required for tRNA translocation from the P-to the E-site (42). This result is in agreement with the finding that 2Ј-deoxy-A 76 -substituted tRNA inhibits ribosomal translocation (43). We have shown that the adenosine at position 76 of tRNA is essential for translocation from the A-to the P-site (Fig. 5). The importance of specific functional groups of tRNA for movement from the A-site to the P-site and from the P-site to the E-site suggests an active ribosomal mechanism for translocation, with essential transient interactions between tRNA and the ribosome.