Transfer RNA-dependent cognate amino acid recognition by an aminoacyl-tRNA synthetase.

Hong, Kwang Won; Ibba, Michael; Weygand-Đurašević, Ivana; Rogers, Michael J.; Thomann, Hans-Ulrich; Söll, Dieter

doi:10.1002/j.1460-2075.1996.tb00549.x

Cited by 50 publications

(39 citation statements)

References 59 publications

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“…As an initial step, we extended this analysis to the determination of individual k cat and K m parameters of the steady-state reaction with respect to glutamine and tRNA Gln (Table 1). These parameters are nearly identical to those determined with the conventional assay, further verifying that this experimental approach is suitable for quantitative kinetic studies (14,23,24).…”

Section: Assay For Steady-state and Single-turnover Aminoacylation Kisupporting

confidence: 72%

“…Further transient kinetics experiments together with simulations and global fitting will be required to derive K d in these circumstances (25,27). Determination of k off for tRNA binding to the unliganded enzyme by fluorescence showed that this parameter is comparable in magnitude to the k chem determined here, consistent with the notion that the tRNA is not in rapid equilibrium (24).…”

Section: Assay For Steady-state and Single-turnover Aminoacylation Kisupporting

confidence: 64%

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Long-range intramolecular signaling in a tRNA synthetase complex revealed by pre-steady-state kinetics

Uter

Perona

2004

Proc. Natl. Acad. Sci. U.S.A.

107

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Pre-steady-state kinetic studies of Escherichia coli glutaminyl-tRNA synthetase conclusively demonstrate the existence of long-distance pathways of communication through the protein-RNA complex. Measurements of aminoacyl-tRNA synthesis reveal a rapid burst of product formation followed by a slower linear increase corresponding to k cat. Thus, a step after chemistry but before regeneration of active enzyme is rate-limiting for synthesis of Gln-tRNA Gln . Single-turnover kinetics validates these observations, confirming that the rate of the chemical step for tRNA aminoacylation (k chem) exceeds the steady-state rate by nearly 10-fold. The concentration dependence of the single-turnover reaction further reveals that the glutamine Kd is significantly higher than the steady-state K m value. The separation of binding from catalytic events by transient kinetics now allows precise interpretation of how alterations in tRNA structure affect the aminoacylation reaction. Mutation of U35 in the tRNA anticodon loop decreases k chem by 30-fold and weakens glutamine binding affinity by 20-fold, demonstrating that the active-site configuration depends on enzyme-tRNA contacts some 40 Å distant. By contrast, mutation of the adjacent G36 has very small effects on k chem and Kd for glutamine. Together with x-ray crystallographic data, these findings allow a comparative evaluation of alternative long-range signaling pathways and lay the groundwork for systematic exploration of how induced-fit conformational transitions may control substrate selection in this model enzyme-RNA complex.R ecognition between proteins and RNA generally occurs by an induced-fit mechanism, in which the protein, the RNA, or both undergo conformational changes en route to the final bound complex (1, 2). Although induced fit necessarily incurs an entropic penalty compared with a preformed and rigid interaction, the built-in flexibility nonetheless may help to increase the kinetic association rate, thus lowering the free-energy barrier for complex formation. In enzymatic reactions, it is also established that induced fit can provide a means to increase substrate specificity: Noncognate substrates may induce partial or incorrect rearrangements, leading to misalignment of reactive moieties along the pathway to the transition state (3). Thus, enzymes that modify RNA may employ induced fit for substrate discrimination at both binding and catalytic steps of the reaction.Aminoacyl-tRNA synthetases are superb model systems for investigating the operation of induced fit in enzyme-RNA complexes. In all organisms, these enzymes maintain fidelity of the genetic code by catalyzing the synthesis of cognate aminoacyl-tRNAs for use in protein synthesis (4). This synthesis occurs by means of a two-step reaction in which the amino acid is first activated to form an aminoacyl adenylate intermediate with release of pyrophosphate (PP i ). In the second step, the nucleophilic oxygen from the 2Ј-OH or 3Ј-OH group on the 3Ј-terminal tRNA ribose sugar attacks the mixed anhydride interm...

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Section: Assay For Steady-state and Single-turnover Aminoacylation Kisupporting

confidence: 72%

Section: Assay For Steady-state and Single-turnover Aminoacylation Kisupporting

confidence: 64%

Long-range intramolecular signaling in a tRNA synthetase complex revealed by pre-steady-state kinetics

Uter

Perona

2004

Proc. Natl. Acad. Sci. U.S.A.

107

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“…A similar discrepancy between k on and k cat /K m was also observed for the hydrolysis reaction catalyzed by the RNA component of RNase P (26), and may indicate the existence of a distinct, nonequilibrating enzyme species formed during the process of product dissociation and subsequent substrate rebinding. Determination of the tRNA off-rate in a binary complex with GlnRS by fluorescence previously yielded k off(tRNA) ϭ 7 s Ϫ1 , suggesting that the affinity for tRNA in the context of a catalytically competent quaternary complex is somewhat weakened relative to a binary complex (27). This is consistent with the need for rapid turnover in the steady state.…”

Section: ϫ1 Smentioning

confidence: 65%

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

Uter

Gruić-Sovulj

Perona

2005

Journal of Biological Chemistry

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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 ϳ60-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 GlnRS⅐tRNA 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.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 (1-3). Accuracy at the first aminoacyl-tRNA synthesis step is accomplished by the aminoacyl-tRNA synthetases, which attach amino acids to the corresponding tRNA species in a two-step reaction. Amino acids are first activated to their corresponding adenylates by harnessing the energy of ATP hydrolysis, generating pyrophosphate. In the second step, the oxygen nucleophile at the 2Ј-or 3Ј-ribose position of the 3Ј-terminal A76 tRNA nucleotide attacks the carbonyl carbon of the mixed anhydride intermediate, forming aminoacyl-tRNA with release of AMP (1). Formation of only cognate aminoacyl-tRNA species requires that the enzymes be specific for both amino acid and tRNA substrates.Although all tRNAs possess largely similar L-shaped tertiary structures, significant capacity for discrimination among these species arises from direct recognition of base-specific functional groups at key nucleotide positions (identity determinants), as well as by an indirect readout process in which selective enzyme interactions are made with groups in the sugar-phosphate backbone or nonspecific portions of the bases (4). The existence of 20 parallel aminoacyl-tRNA systems in vivo promotes further discrimination by competition (5). In contrast, amino acid specificity relies on a much smaller number of possible interactions between the enzyme and substrate; moreover, many of the amino acids possess similar structural and chemical properties. In s...

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“…Although we suggest that this conformation change is important in achieving discrimination, it is likely that other factors are operative as well. It appears that the discrimination process between cognate and noncognate tRNAs is a dynamic one involving conformational changes, substrate binding linkages (Bhattacharyya & Roy, 1993;Hong et al, 1996) and optimization of several functions (Sherman & Soll, 1996).…”

Section: Discussionmentioning

confidence: 99%

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

Mandal¹,

Bhattacharyya²,

Bhattacharyya³

et al. 1998

Protein Science

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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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Transfer RNA-dependent cognate amino acid recognition by an aminoacyl-tRNA synthetase.

Cited by 50 publications

References 59 publications

Long-range intramolecular signaling in a tRNA synthetase complex revealed by pre-steady-state kinetics

Long-range intramolecular signaling in a tRNA synthetase complex revealed by pre-steady-state kinetics

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

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

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