Interactions between tRNA identity nucleotides and their recognition sites in glutaminyl-tRNA synthetase determine the cognate amino acid affinity of the enzyme

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 08/1996; 93(14):6953-8. DOI: 10.1073/pnas.93.14.6953
Source: PubMed


Sequence-specific interactions between aminoacyl-tRNA synthetases and their cognate tRNAs both ensure accurate RNA recognition and prevent the binding of noncognate substrates. Here we show for Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC that the accuracy of tRNA recognition also determines the efficiency of cognate amino acid recognition. Steady-state kinetics revealed that interactions between tRNA identity nucleotides and their recognition sites in the enzyme modulate the amino acid affinity of GlnRS. Perturbation of any of the protein-RNA interactions through mutation of either component led to considerable changes in glutamine affinity with the most marked effects seen at the discriminator base, the 10:25 base pair, and the anticodon. Reexamination of the identity set of tRNA(Gln) in the light of these results indicates that its constituents can be differentiated based upon biochemical function and their contribution to the apparent Gibbs' free energy of tRNA binding. Interactions with the acceptor stem act as strong determinants of tRNA specificity, with the discriminator base positioning the 3' end. The 10:25 base pair and U35 are apparently the major binding sites to GlnRS, with G36 contributing both to binding and recognition. Furthermore, we show that E. coli tryptophanyl-tRNA synthetase also displays tRNA-dependent changes in tryptophan affinity when charging a noncognate tRNA. The ability of tRNA to optimize amino acid recognition reveals a novel mechanism for maintaining translational fidelity and also provides a strong basis for the coevolution of tRNAs and their cognate synthetases.

Download full-text


Available from: Michael Ibba, Dec 23, 2014
  • Source
    • "In particular, unlike most other aaRSs, amino acid recognition and activation by EcGlnRS appear to be tRNA-dependent (43). Consequently, specific GlnRS/tRNAGln interactions affect esterification of tRNAGln and also recognition/activation of the cognate amino acid (44). With these considerations in mind, it was surprising to find that the identity determinants were not conserved in the isoaccepting tRNAsGln of E. coli and yeast cytoplasm and mitochondria (21). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Aminoacylation of transfer RNAGln (tRNAGln) is performed by distinct mechanisms in different kingdoms and represents the most diverged route of aminoacyl-tRNA synthesis found in nature. In Saccharomyces cerevisiae, cytosolic Gln-tRNAGln is generated by direct glutaminylation of tRNAGln by glutaminyl-tRNA synthetase (GlnRS), whereas mitochondrial Gln-tRNAGln is formed by an indirect pathway involving charging by a non-discriminating glutamyl-tRNA synthetase and the subsequent transamidation by a specific Glu-tRNAGln amidotransferase. Previous studies showed that fusion of a yeast non-specific tRNA-binding cofactor, Arc1p, to Escherichia coli GlnRS enables the bacterial enzyme to substitute for its yeast homologue in vivo. We report herein that the same fusion enzyme, upon being imported into mitochondria, substituted the indirect pathway for Gln-tRNAGln synthesis as well, despite significant differences in the identity determinants of E. coli and yeast cytosolic and mitochondrial tRNAGln isoacceptors. Fusion of Arc1p to the bacterial enzyme significantly enhanced its aminoacylation activity towards yeast tRNAGln isoacceptors in vitro. Our study provides a mechanism by which trans-kingdom rescue of distinct pathways of Gln-tRNAGln synthesis can be conferred by a single enzyme.
    Full-text · Article · Jul 2012 · Nucleic Acids Research
  • Source
    • "Gln (CUG) by E. coli glutaminyl-tRNA synthetase (GlnRS) (Ibba et al. 1996), efficient aminoacylation of tRNA 1 "
    [Show abstract] [Hide abstract]
    ABSTRACT: We describe a strategy for tracking Mg²⁺-initiated folding of ³²P-labeled tRNA molecules to their native structures based on the capacity for aminoacylation by the cognate aminoacyl-tRNA synthetase enzyme. The approach directly links folding to function, paralleling a common strategy used to study the folding of catalytic RNAs. Incubation of unfolded tRNA with magnesium ions, followed by the addition of aminoacyl-tRNA synthetase and further incubation, yields a rapid burst of aminoacyl-tRNA formation corresponding to the prefolded tRNA fraction. A subsequent slower increase in product formation monitors continued folding in the presence of the enzyme. Further analysis reveals the presence of a parallel fraction of tRNA that folds more rapidly than the majority of the population. The application of the approach to study the influence of post-transcriptional modifications in folding of Escherichia coli tRNA₁(Gln) reveals that the modified bases increase the folding rate but do not affect either the equilibrium between properly folded and misfolded states or the folding pathway. This assay allows the use of ³²P-labeled tRNA in integrated studies combining folding, post-transcriptional processing, and aminoacylation reactions.
    Full-text · Article · Mar 2012 · RNA
  • Source
    • "Hence, the analysis of unnatural (non-L) amino acids is prohibited. Also large amounts of substrate are required due to the low affinity AARS have toward their cognate amino acid (K M values range of μM to mM) (Eriani et al., 1993; Hill & Schimmel, 1989; Ibba et al., 1996). To offset the accompanying high levels of radioactivity, extremely high amounts of tRNA must be supplied or the concentration of the amino acid must be reduced to levels that are below saturating conditions. "

    Full-text · Chapter · Jun 2011
Show more