Correlation of deformability at a tRNA recognition site and aminoacylation specificity

Abstract

The fidelity of protein synthesis depends on specific tRNA aminoacylation by aminoacyl-tRNA synthetase enzymes, which in turn depends on the recognition of the identity of particular nucleotides and structural features in the substrate tRNA. These features generally reside within the acceptor helix, the anticodon stemloop, and in some systems the variable pocket of the tRNA. In the alanine system, fidelity is ensured by a G⅐U wobble base pair located at the third position within the acceptor helix of alanine tRNA. We have investigated the activity of mutant alanine tRNAs to explore the mechanism of enzyme recognition. Here we show that the mismatched pair C-C is an excellent substitute for G⅐U in alanine-tRNA-knockout cells. A structural investigation by NMR spectroscopy of the C-C RNA acceptor end reveals that the two cytosines are intercalated into the helix, and that C-C exists in multiple conformations. Structural heterogeneity also is present in the wild-type G⅐U RNA, whereas inactive Watson-Crick helices are structurally rigid. The correlation between functional and structural data suggests that the G⅐U pair provides a distinctive structure and a point of deformability that allow the tRNA acceptor end to fit into the active site of the alanyl-tRNA synthetase. Fidelity is ensured because noncognate and inactive mutant tRNAs are bound in the active site in an incorrect conformation that reduces enzymatic activity.A minoacylation of Escherichia coli tRNA Ala by its cognate alanyl-tRNA synthetase (AlaRS) enzyme depends on a G⅐U wobble base pair § in the acceptor helix (Fig. 1); this feature is critical for aminoacylation and can confer partial alanine acceptance on other noncognate tRNAs (1, 2). The features that allow its recognition are likely to include direct interactions with distinctive atomic groups and indirect recognition of the structural and dynamic information encoded in the sequence of the G⅐U pair and its immediate context. The G⅐U wobble base pair provides specific recognition signals in numerous RNAs, ranging from ribosomal RNA and splicing regulatory signals (3) to RNA enzymes (4). A detailed understanding of recognition mechanisms of the G⅐U pair is still lacking. Therefore, tRNA Ala provides not only an important system to understand how fidelity in protein synthesis is ensured, but also a paradigm to dissect the features that allow such a widespread role for the G⅐U pair as a distinctive recognition tag.Here we report the remarkable observation that replacing the G⅐U wobble pair with a C-C mispair preserves tRNA Ala aminoacylation in vivo. An acceptor minihelix RNA containing the C-C substitution has been analyzed by high-resolution NMR spectroscopy and compared with wild-type G⅐U RNA and inactive G⅐C and A⅐U mutant RNAs. The data demonstrate that the two cytosines are intercalated within the acceptor helix, and that C-C exists in multiple conformations in equilibrium on the s-ms time scale. The structure at and around G⅐U and C-C is significantly more mobile than that of Watson-Crick base ...

Structure of the cytosine–cytosine mismatch in the thymidylate synthase mRNA binding site and analysis of its interaction with the aminoglycoside paromomycin

“…In our case, we also did not observe the amino signals in the CC mismatch in both the amino optimized does not reappear at higher paromomycin concentrations, so is not merely a result of a partially occupied binding site, but signal averaging between multiple conformations or motion of the ligand in the binding site. Motion at a CC mismatch was also observed for a mutant tRNA acceptor minihelix (Chang et al 1999) and for a DNA molecule containing a CC mismatch (Boulard et al 1997). It is possible that aminoglycoside binding is linked to or enhanced by the dynamic nature of the CC mismatch.…”

An innate twist between Crick’s wobble and Watson-Crick base pairs

“…It is, therefore, of utmost importance to understand and obtain an estimate of the degree of nonisostericity of nWC base pairs compared to the preponderant WC pairs. It is recognized that the presence of nWC pairs amid WC pairs influences the structure of nucleic acid duplexes, and nonequivalent base-pair substitutions are found to eliminate function (Chang et al 1999;Zhong et al 2006), thus playing a major role in the evolution of macromolecules (Gutell et al 1994). However, rationale for the observed nWC base pair-mediated mechanistic effects in RNA duplexes still remains vague.…”

Dual Mode Recognition of Two Isoacceptor tRNAs by Mammalian Mitochondrial Seryl-tRNA Synthetase

“…These findings clearly indicated that the D loop-T⌿C loop tertiary interactions are required for the serylation of mt tRNA UGA Ser , whereas only the sequence of the T⌿C loop is important for the activity of mt tRNA GCU Ser . Aminoacylation of E. coli tRNA Ala by alanyl-tRNA synthetase (AlaRS) depends on a G3⅐U70 wobble base pair in the acceptor stem (37). Understanding such a unique identity determinant was greatly advanced by the demonstration that the minihelix variant, composed only of the amino acid acceptor-T⌿C helix, could be a good substrate for AlaRS (38, 39).…”