Contributions of discrete tRNA<sup>Ser</sup>domains to aminoacylation by<i>E.coli</i>seryl-tRNA synthetase: a kinetic analysis using model RNA substrates

Sampson, Jeffrey R.; Saks, Margaret E.

doi:10.1093/nar/21.19.4467

Cited by 84 publications

(96 citation statements)

References 36 publications

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“…Similar to several other aaRSs (19)(20)(21), the PP i product generated in the reaction was found to be a potent inhibitor of AlaRS in vitro, and the presence of inorganic pyrophosphatase (PPase) dramatically increased the reaction rate under many reaction conditions. In addition, the initial rates and levels of aminoacylation of several tRNA Ala identity mutations are differentially stimulated by the presence of PPase.…”

mentioning

confidence: 65%

“…Because it is likely that all tRNA synthetases to some degree are inhibited by PP i , in vitro experiments defining tRNA recognition nucleotides should be reexamined for PPase effects. In the case of E. coli seryl-tRNA synthetase, PPase was found to uniformly increase k cat ͞K M of all substrates tested (20). Because tRNA Catalytic efficiency loss is calculated as the ratio of (kcat͞KM)YFA2͞(kcat͞KM)varian.…”

Section: Discussionmentioning

confidence: 98%

See 1 more Smart Citation

Modulation of tRNA ^Ala identity by inorganic pyrophosphatase

Wolfson

Uhlenbeck

2002

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

124

163

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A highly sensitive assay of tRNA aminoacylation was developed that directly measures the fraction of aminoacylated tRNA by following amino acid attachment to the 3 -32 P-labeled tRNA. When applied to Escherichia coli alanyl-tRNA synthetase, the assay allowed accurate measurement of aminoacylation of the most deleterious mutants of tRNA Ala . The effect of tRNA Ala identity mutations on both aminoacylation efficiency (kcat͞KM) and steady-state level of aminoacyl-tRNA was evaluated in the absence and presence of inorganic pyrophosphatase and elongation factor Tu. Significant levels of aminoacylation were achieved for tRNA mutants even when the kcat͞KM value is reduced by as much as several thousandfold. These results partially reconcile the discrepancy between in vivo and in vitro analysis of tRNA Ala identity.T wo major approaches have been developed to identify nucleotides required for tRNA recognition by aminoacyl-tRNA (aa-tRNA) synthetases (aaRSs). The first involves introducing a suppressor anticodon into a tRNA of interest and then examining the suppressor activity of the point mutations in vivo (1). The second involves assaying aminoacylation activity of mutant tRNAs made by transcription in vitro (2). Both methods reveal a limited number of nucleotides critical for aminoacylation, the importance of which was confirmed by performing ''swap'' experiments in which the proposed nucleotides were transplanted into the body of another tRNA (2, 3). Subsequent analysis of aminoacylation rate in vitro or sequencing of the reporter proteins in vivo was used to confirm that change in tRNA identity had occurred. These two methods generally agree reasonably well with each other and correlate well with observed base-specific contacts in cocrystal structures of tRNA-aaRS complexes (4-6). However, there are several examples where the conclusions derived from in vitro and in vivo approaches disagree, the most notable of which is the recognition of tRNA Ala by Escherichia coli alanyl-tRNA synthetase (AlaRS). Although no cocrystal structure is available, in vitro experiments with truncated RNA substrates (7, 8) and mutant tRNA Ala (9) as well as identity-swap experiments (10, 11) strongly suggested that the G3⅐U70 pair is the major identity element of tRNA Ala . The deleterious effect of mutations of this base pair significantly exceeds those of other tRNA Ala recognition elements (8, 9, 12-15) and clearly was indicative of the predominant role of this base pair and in particular the exocyclic amino group of G3 in recognition. In contrast, in vivo experiments showed that many of same mutations in the G3⅐U70 base pair have only a moderate effect on the levels of aminoacylated tRNA Ala in E. coli and consequently on the ability of the mutant tRNAs to function in protein synthesis (16,17). A careful analysis of the in vivo experiments indicates that the observed discrepancy was not caused by the technical problems associated with the determining of tRNA aminoacylation levels in vivo, and therefore it was proposed that the discre...

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confidence: 65%

Section: Discussionmentioning

confidence: 98%

Modulation of tRNA ^Ala identity by inorganic pyrophosphatase

Wolfson

Uhlenbeck

2002

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

124

163

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“…Support of such a notion comes from the fact that high conservation of nucleotides in this region of bacterial tRNAs Ser cannot be detected; contrary to that, the G30:C40 base pair is absolutely conserved among archaeal tRNA Ser species. This position has been identified as an identity determinant for human phenylalanyl-tRNA synthetase (40 tRNA Ser Recognition in M. barkeri 48782 tors, it makes the largest contribution to serine identity, as confirmed by different experimental approaches (9,17,41). Despite direct contacts that have been observed between T. thermophilus SerRS and the variable arm stem (7), sequence alterations of the variable arm only insignificantly affected the serylation efficiency (27).…”

Section: Barkeri Possesses Two Functional Serrs Enzymes-bothmentioning

confidence: 87%

Differential Modes of Transfer RNASer Recognition in Methanosarcina barkeri

Korenčić

Polycarpo

Weygand-Đurašević

et al. 2004

Journal of Biological Chemistry

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Two dissimilar seryl-transfer RNA (tRNA) synthetases (SerRSs) exist in Methanosarcina barkeri, one of bacterial type and the other resembling SerRSs present only in some methanogenic archaea. To investigate the requirements of these enzymes for tRNA Ser recognition, serylation of variant transcripts of M. barkeri tRNA Ser was kinetically analyzed in vitro with pure enzyme preparations. Characteristically for the serine system, the length of the variable arm was shown to be crucial for both enzymes, as was the identity of the discriminator base (G73). Moreover, a novel determinant for the specific tRNA Ser recognition was identified as the anticodon stem base pair G30:C40; its contribution to the efficiency of serylation was remarkable for both SerRSs. However, despite these similarities, the two SerRSs do not possess a uniform mode of tRNA Ser recognition, and additional determinants are necessary for serylation specificity by the methanogenic enzyme. In particular, the methanogenic SerRS relies on G1:C72 identity and on the number of unpaired nucleotides at the base of the variable stem for tRNA Ser recognition, unlike its bacterial type counterpart. We propose that such a distinction between the two enzymes in tRNA Ser identity determinants reflects their evolutionary pathways, hence attesting to their diversity.To maintain translational accuracy, aminoacyl-transfer RNA (tRNA) 1 synthetases are highly selective toward their amino acid and tRNA substrates. In the process of tRNA recognition, the cognate and non-cognate substrates are discriminated according to characteristic nucleotides in certain positions of the tRNA, specific for the tRNA/synthetase system.

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“…32 P]-UTP-labeled RNAs for elution experiments detailed in Figure 1A and for UV cross-linking were generated as previously described using SP6 RNA polymerase (Macdonald et al+, 1995)+ Unlabeled 3ϫSREϩ and 3ϫSREϪ RNAs were generated using T7 RNA polymerase as described by Sampson & Saks (1993) with the exception that GMP was omitted from the reaction+…”

Section: Generation Of Sre Rnasmentioning

confidence: 99%