Functional group recognition at the aminoacylation and editing sites of E. coli valyl-tRNA synthetase.

To correct misactivation and misacylation errors, Escherichia coli valyl-tRNA synthetase (ValRS) catalyzes a tRNA(Val)-dependent editing reaction at a site distinct from its aminoacylation site. Here we examined the effects of replacing the conserved 3'-adenosine of tRNA(Val) with nucleoside analogs, to identify structural elements of the 3'-terminal nucleoside necessary for tRNA function at the aminoacylation and editing sites of ValRS. The results show that the exocyclic amino group (N6) is not essential: purine riboside-substituted tRNA(Val) is active in aminoacylation and in stimulating editing. Presence of an O6 substituent (guanosine, inosine, xanthosine) interferes with aminoacylation as well as posttransfer and total editing (pre- plus posttransfer editing). Because ValRS does not recognize substituents at the 6-position, these results suggest that an unprotonated N1, capable of acting as an H-bond acceptor, is an essential determinant for both the aminoacylation and editing reactions. Substituents at the 2-position of the purine ring, either a 2-amino group (2-aminopurine, 2,6-diaminopurine, guanosine, and 7-deazaguanosine) or a 2-keto group (xanthosine, isoguanosine), strongly inhibit both aminoacylation and editing. Although aminoacylation by ValRS is at the 2'-OH, substitution of the 3'-terminal adenosine of tRNA(Val) with 3'-deoxyadenosine reduces the efficiency of valine acceptance and of posttransfer editing, demonstrating that the 3'-terminal hydroxyl group contributes to tRNA recognition at both the aminoacylation and editing sites. Our results show a strong correlation between the amino acid accepting activity of tRNA and its ability to stimulate editing, suggesting misacylated tRNA is a transient intermediate in the editing reaction, and editing by ValRS requires a posttransfer step.

Transfer RNA determinants for translational editing by Escherichia coli valyl-tRNA synthetase.

Valyl-tRNA synthetase (ValRS) has difficulty differentiating valine from structurally similar non-cognate amino acids, most prominently threonine. To minimize errors in aminoacylation and translation the enzyme catalyzes a proofreading (editing) reaction that is dependent on the presence of cognate tRNA(Val). Editing occurs at a site functionally distinct from the aminoacylation site of ValRS and previous results have shown that the 3'-terminus of tRNA(Val) is recognized differently at the two sites. Here, we extend these studies by comparing the contribution of aminoacylation identity determinants to productive recognition of tRNA(Val) at the aminoacylation and editing sites, and by probing tRNA(Val) for editing determinants that are distinct from those required for aminoacylation. Mutational analysis of Escherichia coli tRNA(Val) and identity switch experiments with non-cognate tRNAs reveal a direct relationship between the ability of a tRNA to be aminoacylated and its ability to stimulate the editing activity of ValRS. This suggests that at least a majority of editing by the enzyme entails prior charging of tRNA and that misacylated tRNA is a transient intermediate in the editing reaction.

Functional importance of the 3'-terminal adenosine of tRNA in ribosomal translation.

The universally conserved 3'-terminal CCA sequence of tRNA interacts with large ribosomal subunit RNA during translation. The functional importance of the interaction between the 3'-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 message. 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 peptide bond formation, although the donor activity of the mutant NAcVal-tRNA(CCG) in the peptidyl transfer reaction is slightly reduced compared with wild-type NAcVal-tRNA. Translocation of 3'-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 3'-terminal nucleotide of tRNA is critical for tRNA movement in the ribosome.