Anticodon and acceptor stem nucleotides in tRNAGln are major recognition elements for E. coli glutaminyl-tRNA synthetase

Jahn, Martina; Mj, Rogers; Söll, Dieter

doi:10.1038/352258a0

Cited by 207 publications

(217 citation statements)

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“…The conformational rearrangements converge in the active-site domain, where portions of both the amino acidand ATP-binding sites are misoriented with respect to each other in the absence of tRNA (9,11,12). Because the tRNA also is rearranged compared with its presumed solution structure, formation of the GlnRS-tRNA complex is best described as a mutual induced fit involving conformational changes of both macromolecular partners.Several studies have implicated nucleotides in the anticodon loop and acceptor end as GlnRS specificity determinants (13)(14)(15). A peptide loop binding the extreme inner corner of the tRNA L-shape and a ␤-ribbon emanating from a distal ␤-barrel domain are identified as potential mediators of pathways by which the distal tRNA contacts may be communicated to the active site (16,17).…”

mentioning

confidence: 99%

“…Several studies have implicated nucleotides in the anticodon loop and acceptor end as GlnRS specificity determinants (13)(14)(15). A peptide loop binding the extreme inner corner of the tRNA L-shape and a ␤-ribbon emanating from a distal ␤-barrel domain are identified as potential mediators of pathways by which the distal tRNA contacts may be communicated to the active site (16,17).…”

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confidence: 99%

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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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confidence: 99%

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confidence: 99%

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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“…The tRNAG'" induces a local conformational change that may contribute substantially to the discrimination. This tRNAG'"-induced conformational change is likely to be related to the conformational changes implicated in the Vun't HoffpZot studies of SOH and co-workers (Jahn et al, 1991;Sherman et al, 1996), although the precise relationships remains to be elucidated. Although we suggest that this conformation change is important in achieving discrimination, it is likely that other factors are operative as well.…”

Section: Discussionmentioning

confidence: 88%

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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“…This sequence conservation may be involved in the contribution to discrimination for non-cognate aminoacyl-tRNA synthetase. The occurrence of C2-G71 in tRNA Thr would be useful in precluding tRNA Thr from being mischarged by several noncognate aminoacyl-tRNA synthetases that recognize the second base pair other than the C2-G71 of the corresponding tRNAs, for example, in E. coli, glutaminyl- [29], glutamyl- [30], and methionyl- [31] tRNA synthetases.…”

Section: Acceptor Stemmentioning

confidence: 99%

Identity elements of Thermus thermophilus tRNA^Thr

1996

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In this study, we identified nucleotides that specify aminoacylation of tRNA TM by Thermus thermophilus threonyltRNA synthetase (ThrRS) using in vitro transcripts. Mutation studies showed that the first base pair in the acceptor stem as well as the second and third positions of the anticodon are major identity elements of T. thermophilus tRNA TM, which are essentially the same as those of Escherichia coli tRNA TM. The discriminator base, U73, also contributed to the specific aminoacylation, but not the second base pair in the acceptor stem. These findings are in contrast to E. coli tRNA TM, where the second base pair is required for threonylation, with the discriminator base, A73, playing no roles. In addition, among several mutations at the third base pair in the acceptor stem, only the G3-U70 mutant was a poor substrate for ThrRS, suggesting that the G3-UTo wobble pair, which is the identity determinant of tRNA Aj", acts as a negative element for ThrRS. Similar results were obtained in E. coli and yeast. Thus, this manner of rejection of tRNA Ala is also likely to have been retained in the threonine system throughout evolution.

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Anticodon and acceptor stem nucleotides in tRNAGln are major recognition elements for E. coli glutaminyl-tRNA synthetase

Cited by 207 publications

References 32 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

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

Identity elements of Thermus thermophilus tRNA^Thr

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Anticodon and acceptor stem nucleotides in tRNAGln are major recognition elements for E. coli glutaminyl-tRNA synthetase

Cited by 207 publications

References 32 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

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

Identity elements of Thermus thermophilus tRNAThr

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Identity elements of Thermus thermophilus tRNA^Thr