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: 9.81). 08/1996; 93(14):6953-8. DOI: 10.1073/pnas.93.14.6953
Source: PubMed

ABSTRACT 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
    • "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.
    RNA 03/2012; 18(3):569-80. DOI:10.1261/rna.030080.111 · 4.62 Impact Factor
  • Source
    • "The molecular mechanism for this tRNA requirement has been elucidated for GluRS, which binds ATP in its active site in the absence of tRNA Glu , but does so in such a way that the two reacting groups in the glutamate activation reaction, the a-phosphate of ATP and the a-COOH of glutamate, are too far apart for glutamate activation to occur [10]; in the presence of tRNA Glu , ATP binds in another orientation which allows glutamate activation. In the case of GlnRS, and even in the case of tryptophanyl-tRNA synthetase (TrpRS) which can activate its amino acid substrate in the absence of tRNA, the tRNA identity elements influence the binding of the amino acid substrate [15], and therefore alter directly or indirectly the structure of the active site. Moreover, the structure of an inactive complex between yeast tRNA Asp and E. coli AspRS suggests that the acceptor stem controls the proper positioning of the amino acid substrate [16]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Asparaginyl-tRNA formation in Pseudomonas aeruginosa PAO1 involves a nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) which forms Asp-tRNAAsp and Asp-tRNAAsn, and a tRNA-dependent amidotransferase which transamidates the latter into Asn-tRNAAsn. We report here that the inhibition of this ND-AspRS by L-aspartol adenylate (Asp-ol-AMP), a stable analog of the natural reaction intermediate L-aspartyl adenylate, is biphasic because the aspartylation of the two tRNA substrates of ND-AspRS, tRNAAsp and tRNAAsn, are inhibited with different Ki values (41 μM and 215 μM, respectively). These results reveal that the two tRNA substrates of ND-AspRS interact differently with its active site. Yeast tRNAAsp transcripts with some identity elements replaced by those of tRNAAsn have their aspartylation inhibited with Ki values different from that for the wild-type transcript. Therefore, aminoacyl adenylate analogs, which are competitive inhibitors of their cognate aminoacyl-tRNA synthetase, can be used to probe rapidly the role of various structural elements in positioning the tRNA acceptor end in the active site.
    Journal of Enzyme Inhibition and Medicinal Chemistry 10/2008; 22(1). · 2.38 Impact Factor
  • Source
    • "Abbreviations: GlnRS, glutaminyl-tRNA synthetase; acrylodan, 6-acryloyl- 2-dimethyl aminonaphthalene; CD, circular dichroism: tempol, (4-hydroxy- 2,2,6,6-tetramethylpiperidine-I -0xyl): GluRS, glutamyl-tRNA synthetase. of the best systems to study the recognition and the consequent catalytic activation process. Jahn et al. (1991) and Ibba et al. (1996) have shown that the correct recognition of anti-codon bases increases kc,, of the enzyme glutaminyl-tRNA synthetase of Escherichia coli significantly. Because the anti-codon binding pocket is approximately 35 A away from the active site, a protein conformational change or a tRNA conformational change or both are responsible for transmitting the anti-codon binding information to the active site. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Conformational changes that occur upon substrate binding are known to play crucial roles in the recognition and specific aminoacylation of cognate tRNA by glutaminyl-tRNA synthetase. In a previous study we had shown that glutaminyl-tRNA synthetase labeled selectively in a nonessential sulfhydryl residue by an environment sensitive probe, acrylodan, monitors many of the conformational changes that occur upon substrate binding. In this article we have shown that the conformational change that occurs upon tRNA(Gln) binding to glnRS/ATP complex is absent in a noncognate tRNA tRNA(Glu)-glnRS/ATP complex. CD spectroscopy indicates that this cognate tRNA(Gln)-induced conformational change may involve only a small change in secondary structure. The Van't Hoff plot of cognate and noncognate tRNA binding in the presence of ATP is similar, suggesting similar modes of interaction. It was concluded that the cognate tRNA induces a local conformational change in the synthetase that may be one of the critical elements that causes enhanced aminoacylation of the cognate tRNA over the noncognate ones.
    Protein Science 04/2008; 7(4):1046-51. DOI:10.1002/pro.5560070422 · 2.85 Impact Factor
Show more