A cognate tRNA specific conformational change in glutaminyl‐tRNA synthetase and its implication for specificity

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 glutaminyltRNA 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 tRNAG'" binding to glnRS/ATP complex is absent in a noncognate tRNA tRNAG1"-glnRS/ATP complex. CD spectroscopy indicates that this cognate tRNAG1"-induced conformational change may involve only a small change in secondary structure. The Van? 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.Keywords: conformational change; discrimination; fluorescence; synthetase: tRNA Translation of nucleotide sequences in mRNAs to amino acid sequences in proteins is characterized by high degree of accuracy. This level of accuracy is primarily determined at the level of aminoacylation of tRNAs by aminoacyl-tRNA synthetases (Schimme1 & Soll, 1979;Schimmel, 1987;Carter, 1993). The correct aminoacylation of all the tRNAs involves positive recognition of the cognate tRNAs (McClain, 1993) and negative discrimination of the noncognate tRNAs (Schmitt et al., 1993). Recognition of cognate tRNAs has been studied intensively, and structural elements that are responsible for recognition have now been mapped for many systems (Normanly & Abelson, 1989;Mechulam et al., 1995). Very little is known, however, about the factors that are responsible for negative discrimination.Even in the case of recognition of cognate tRNAs where the identity elements have been mapped, it is not clear exactly how this elements influence enzymatic properties and translates recognition into catalytic competence. Due to pioneering work by Soll and co-workers, glutaminyl-tRNA synthetase has emerged as one Reprint requests to: Siddhartha Roy, Department of Biophysics, Bose Institute, P 1/12. C.I.T. Scheme VI1 M, Calcutta 700054, India; e-mail: siddarth@boseinst.ernet.in.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 ...

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