Amino Acid-dependent Transfer RNA Affinity in a Class I Aminoacyl-tRNA Synthetase

Nathan T Uter,Ita Gruic-Sovulj,John J Perona

doi:10.1074/jbc.m414259200

Nathan T Uter, Ita Gruic-Sovulj + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m414259200

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Abstract

Steady-state and transient kinetic analyses of glutaminyl-tRNA synthetase (GlnRS) reveal that the enzyme discriminates against noncognate glutamate at multiple steps during the overall aminoacylation reaction. A major portion of the selectivity arises in the amino acid activation portion of the reaction, whereas the discrimination in the overall two-step reaction arises from very weak binding of noncognate glutamate. Further transient kinetics experiments showed that tRNA(Gln) binds to GlnRS approximately 60-fold weaker when noncognate glutamate is present and that glutamate reduces the association rate of tRNA with the enzyme by 100-fold. These findings demonstrate that amino acid and tRNA binding are interdependent and reveal an important additional source of specificity in the aminoacylation reaction. Crystal structures of the GlnRS x tRNA complex bound to either amino acid have previously shown that glutamine and glutamate bind in distinct positions in the active site, providing a structural basis for the amino acid-dependent modulation of tRNA affinity. Together with other crystallographic data showing that ligand binding is essential to assembly of the GlnRS active site, these findings suggest a model for specificity generation in which required induced-fit rearrangements are significantly modulated by the identities of the bound substrates.

Highlights

The high fidelity of protein synthesis in living cells arises as a consequence of specificity at three distinct steps in the pathway: aminoacyl-tRNA formation, selection of aminoacyltRNA by elongation factor Tu, and ribosomal proofreading of the codon-anticodon interaction [1,2,3]
Discrimination against Glutamate at the Amino Acid Activation Step—To evaluate the contribution of the aminoacyl adenylate synthesis step to the overall accuracy of glutaminylation, the rate of exchange of 32P-labeled pyrophosphate into ATP was measured in the presence of either cognate glutamine or noncognate glutamate (Table I)
Unlike most tRNA synthetases, glutaminyl-tRNA synthetase (GlnRS) does not synthesize the aminoacyl adenylate intermediate in the absence of tRNA

Summary

The abbreviations used are

GluRS, glutamyl-tRNA synthetase; GlnRS, glutaminyl-tRNA synthetase; DTT, dithiothreitol; QSI, 5Ј-O-[N(L-glutaminyl)sulfamoyl] adenosine; Mes, 4-morpholineethanesulfonic acid; Pipes, 1,4-piperazinediethanesulfonic acid; AMPCPP, adenosine 5Ј-(␣,␤-methylene)triphosphate. To explore the detailed origins of amino acid specificity in GlnRS, we performed steady-state and pre-steady-state kinetic studies utilizing both the conventional ATP-PPi exchange reaction, as well as a newer assay that relies upon internal 32P labeling of tRNA [16] This assay is highly sensitive and permits use of very high amino acid concentrations, a requirement for rigorous measurements of noncognate reactions in which ligand affinities are weak. The presteady-state kinetic analysis uncovers a significant coupling between the amino acid and tRNA affinities of the enzyme, in which the identity of the bound amino acid (cognate or noncognate) controls how tightly tRNAGln binds This finding suggests that induced-fit structural pathways connecting the amino acid and tRNA binding sites may contribute to enzyme specificity. The common presence of induced-fit rearrangements among many tRNA synthetases, as revealed by x-ray structures in the apo- and liganded states, suggests that this mechanism may be quite general to the enzyme family

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DISCUSSION