Uniform Binding of Aminoacylated Transfer RNAs to the Ribosomal A and P Sites

Fahlman, Richard P.; Dale, Taraka; Uhlenbeck, Olke C.

doi:10.1016/j.molcel.2004.10.030

Cited by 93 publications

(129 citation statements)

References 57 publications

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“…in determining the rate of decoding (38,39). In the above experiments, peptidyl-tRNAs differed not only in the C-terminal amino acid, but in the nature of the tRNA as well.…”

Section: Rate Constants Of Pmn Reaction With Differentmentioning

confidence: 86%

Modulation of the Rate of Peptidyl Transfer on the Ribosome by the Nature of Substrates

Wohlgemuth

Brenner

Beringer

et al. 2008

Journal of Biological Chemistry

152

194

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The ribosome catalyzes peptide bond formation between peptidyl-tRNA in the P site and aminoacyl-tRNA in the A site. Here, we show that the nature of the C-terminal amino acid residue in the P-site peptidyl-tRNA strongly affects the rate of peptidyl transfer. Depending on the C-terminal amino acid of the peptidyl-tRNA, the rate of reaction with the small A-site substrate puromycin varied between 100 and 0.14 s ؊1 , regardless of the tRNA identity. The reactivity decreased in the order Lys ‫؍‬ Arg > Ala > Ser > Phe ‫؍‬ Val > Asp Ͼ Ͼ Pro, with Pro being by far the slowest. However, when Phe-tRNA Phe was used as A-site substrate, the rate of peptide bond formation with any peptidyl-tRNA was ϳ7 s ؊1 , which corresponds to the rate of binding of Phe-tRNA Phe to the A site (accommodation). Because accommodation is rate-limiting for peptide bond formation, the reaction rate is uniform for all peptidyltRNAs, regardless of the variations of the intrinsic chemical reactivities. On the other hand, the 50-fold increase in the reaction rate for peptidyl-tRNA ending with Pro suggests that full-length aminoacyl-tRNA in the A site greatly accelerates peptide bond formation.The enzymatic activity of the ribosome is to catalyze peptide bond formation. During the peptidyl transfer reaction, the ␣-amino group of aminoacyl-tRNA bound to the A site of the ribosome attacks the ester bond of peptidyl-tRNA in the P site, which results in peptidyl-tRNA extended by one amino acid in the A site and deacylated tRNA in the P site. The tRNA substrates are aligned in the active center of the ribosome by interactions of their CCA ends with 23 S rRNA bases (1-3). The ribosome lowers the activation entropy of the reaction (4, 5) by orienting the reacting groups precisely relative to each other (2, 3), providing an electrostatic environment that reduces the free energy of forming the transition state, shielding the reaction against bulk water (6, 7), or a combination of these effects (8).The peptidyl transfer reaction is modulated by conformational changes at the active site (3, 8 -10) as well as by the nature of the substrates. Rapid peptide bond formation requires fulllength tRNA in both A and P sites, and the reaction rate is influenced by the length of the tRNA fragments when model substrates are used (8, 10 -14). The reaction rate is also influenced by the nature of the amino acid side chain of the A-site substrate (13,(15)(16)(17), but is independent of the nucleophilicity of the attacking amino group in model substrates (18). Moreover, the length of the peptidyl chain and the nature of the C-terminal amino acid of the peptidyl-tRNA in the P site seem to have an effect (10,12,13,19). Early studies with 50 S ribosomal subunits indicated that efficient peptidyl transfer was observed with 3Ј-terminal RNase T1 fragments of N-acetylArg-tRNA Arg and fMet-tRNA fMet as model P-site substrates and an analog of aminoacyl-tRNA, puromycin (Pmn 4 ; O-methyltyrosine linked to N 6 -dimethyladenosine via an amide bond), as A-site substrate (20). In contrast,...

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“…in determining the rate of decoding (38,39). In the above experiments, peptidyl-tRNAs differed not only in the C-terminal amino acid, but in the nature of the tRNA as well.…”

Section: Rate Constants Of Pmn Reaction With Differentmentioning

confidence: 86%

Modulation of the Rate of Peptidyl Transfer on the Ribosome by the Nature of Substrates

Wohlgemuth

Brenner

Beringer

et al. 2008

Journal of Biological Chemistry

152

194

View full text Add to dashboard Cite

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“…Tight-coupled 70S ribosomes from E. coli MRE600 cells were prepared as described (44) and purified pellets were resuspended in buffer RB (50 mM HEPES [pH 7.0], 30 mM KCl, 70 mM NH 4 Cl, 10 mM MgCl 2 , and 1 mM DTT). Ribosomes were flash frozen and stored at −80°C and activated as previously described (45). mRNA derivatives of the T4 gp32 mRNA fragment 5′-GGCAAGGAGGUAAAAAUGXXXGCACGU-3′, where XXX indicates the A site codon, were purchased from Dharmacon.…”

Section: Methodsmentioning

confidence: 99%

Tuning the affinity of aminoacyl-tRNA to elongation factor Tu for optimal decoding

Schrader

Chapman

Uhlenbeck

2011

Proc. Natl. Acad. Sci. U.S.A.

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123

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To better understand why aminoacyl-tRNAs (aa-tRNAs) have evolved to bind bacterial elongation factor Tu (EF-Tu) with uniform affinities, mutant tRNAs with differing affinities for EF-Tu were assayed for decoding on Escherichia coli ribosomes. At saturating EF-Tu concentrations, weaker-binding aa-tRNAs decode their cognate codons similarly to wild-type tRNAs. However, tighter-binding aa-tRNAs show reduced rates of peptide bond formation due to slow release from EF-Tu•GDP. Thus, the affinities of aa-tRNAs for EF-Tu are constrained to be uniform by their need to bind tightly enough to form the ternary complex but weakly enough to release from EF-Tu during decoding. Consistent with available crystal structures, the identity of the esterified amino acid and three base pairs in the T stem of tRNA combine to define the affinity of each aa-tRNA for EF-Tu, both off and on the ribosome.T he ternary complex of bacterial elongation factor Tu (EF-Tu), GTP and aminoacyl-tRNA (aa-tRNA) binds to the ribosome and participates in a multistep decoding pathway in which GTP is hydrolyzed, EF-Tu•GDP is released, and the aa-tRNA enters the ribosomal A site (1-6). Although all elongator aa-tRNAs bind EF-Tu•GTP with similar affinities (7-9), studies with misacylated tRNAs reveal that the protein shows substantial specificity for both the esterified amino acid and the tRNA body (10-12). The nearly uniform EF-Tu binding affinity observed for tRNAs acylated with their correct (cognate) amino acid occurs because the sequence of each tRNA has evolved to compensate for the variable thermodynamic contribution of the esterified amino acid. Thus, weak-binding esterified amino acids such as glycine and alanine have corresponding tRNAs that bind the protein tightly, while tight-binding amino acids such as tyrosine or glutamine have corresponding tRNAs that bind poorly. The crystal structure of Thermus aquaticus EF-Tu•GTP bound to Saccharomyces cerevisiae Phe-tRNA Phe (13) reveals that the protein primarily forms extensive interactions with the helical phosphodiester backbone of the acceptor and T stems of tRNA Phe . Recent protein (14) and tRNA (15, 16) mutagenesis experiments indicate that much of the specificity is the result of interactions made between three amino acids of EF-Tu and three adjacent base pairs in the T stem (16). Additional mutagenesis experiments indicate that the thermodynamic contribution of each of the three base pairs is independent of the others, making it possible to adjust the affinity of aa-tRNAs to EF-Tu in a predictable manner.Although a detailed structural and thermodynamic understanding of how EF-Tu achieves uniform binding with different aa-tRNAs is beginning to emerge, the underlying selective pressures that lead to uniform binding are less clear. While aa-tRNAs must bind EF-Tu tightly enough to participate in translation, the high intracellular concentration of EF-Tu (17) ensures that they do not significantly compete with one another for the protein.It therefore seems unlikely that a minimum threshold binding af...

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“…Tightly coupled 70S ribosomes from exponential E. coli MRE600 were purified, stored in small aliquots in 50 mM HEPES (pH 7.5), 10 mM MgCl 2 , 70 mM NH 4 Cl, 1 mM DTT buffer at −80°C, and heat activated before reaction, as previously described (Fahlman et al 2004). E. coli EF-Tu was purified from a His6-TEV expression clone (Chapman et al 2012).…”

Section: Methodsmentioning

confidence: 99%

“…Unmodified E. coli tRNA Ala GGC and its mutants were prepared by in vitro transcription by T7 RNA polymerase from templates generated by primer extension of overlapping DNA oligonucleotides (IDT) and purified via 10% denaturing PAGE. Native tRNA Ala was isolated from unfractionated E. coli tRNA using an oligonucleotide hybridization protocol (Fahlman et al 2004). E. coli tRNA fMet was purchased from SigmaAldrich.…”

Section: Methodsmentioning

confidence: 99%

tRNA residues evolved to promote translational accuracy

Shepotinovskaya

Uhlenbeck

2013

RNA

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The decoding properties of 22 structurally conservative base-pair and base-triple mutations in the anticodon hairpin and tertiary core of Escherichia coli tRNA Ala GGC were determined under single turnover conditions using E. coli ribosomes. While all of the mutations were able to efficiently decode the cognate GCC codon, many showed substantial misreading of near-cognate GUC or ACC codons. Although all the misreading mutations were present in the sequences of other E. coli tRNAs, they were never found among bacterial tRNA Ala GGC sequences. This suggests that the sequences of bacterial tRNA Ala GGC have evolved to avoid reading incorrect codons.

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Uniform Binding of Aminoacylated Transfer RNAs to the Ribosomal A and P Sites

Cited by 93 publications

References 57 publications

Modulation of the Rate of Peptidyl Transfer on the Ribosome by the Nature of Substrates

Modulation of the Rate of Peptidyl Transfer on the Ribosome by the Nature of Substrates

Tuning the affinity of aminoacyl-tRNA to elongation factor Tu for optimal decoding

tRNA residues evolved to promote translational accuracy

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