A new assay for tRNA aminoacylation kinetics

Wolfson, Alexey D.; Pleiss, Jeffrey A.; Uhlenbeck, Olke C.

doi:10.1017/s1355838298980700

Cited by 56 publications

(62 citation statements)

References 24 publications

(19 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…When measured in the tRNA aminoacylation reaction, the K M for Ala is 47 M in the absence and 8 M in the presence of PPase. These data agree well with a previous observation that an increase in Ala concentration from 10 M to 1 mM translated in only a 3-fold increase in aminoacylation rate (23). A similar K M for Ala (13 M) in the presence of PPase was obtained by using the traditional assay.…”

Section: Resultssupporting

confidence: 92%

“…We previously designed an assay using an acid polyacrylamide gel to follow the aminoacylation of 3Ј-32 P-labeled tRNA with nonradioactive amino acid (23). Although this assay had high sensitivity and directly measured the fraction of aa-tRNA, it was labor-intensive, and the gel-separation step generally required the use of bimolecular tRNAs substrates with a nick in T-stem, limiting its universality.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Modulation of tRNA ^Ala identity by inorganic pyrophosphatase

Wolfson

Uhlenbeck

2002

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

Self Cite

123

163

View full text Add to dashboard Cite

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...

show abstract

Section: Resultssupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Modulation of tRNA ^Ala identity by inorganic pyrophosphatase

Wolfson

Uhlenbeck

2002

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

Self Cite

123

163

View full text Add to dashboard Cite

show abstract

“…The reaction was conducted in a rapid chemical-quench instrument using 71 μM GlnRS, 33 μM [ 32 P]-labeled tRNA Gln , 2 M glutamate, and 10 mM ATP. The quenched time points of the reaction were analyzed according to Wolfson-Uhlenbeck method [56]. The ratio of Glu~AMP and AMP gives the fraction aminoacylated at each timepoint.…”

Section: Discussionmentioning

confidence: 99%

“…(If product is quantitated as radiolabeled AA-tRNA AA , those amino acids not available as radionuclides cannot, of course, be employed as substrates.) These limitations are overcome by a novel assay that employs 3′-[ 32 P]-labeled tRNA together with unlabeled amino acid [56,57]. The assay (described more fully in the contribution of Uhlenbeck, this volume) has been used to determine both steady-state and microscopic kinetic constants, and should be widely applicable to most if not all aminoacyl-tRNA synthetases.…”

Section: The Steady State Aminoacylation Reactionmentioning

confidence: 99%

Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases

Francklyn

First

Perona

et al. 2008

Methods

105

120

View full text Add to dashboard Cite

The accuracy of protein synthesis relies on the ability of aminoacyl-tRNA synthetases (aaRSs) to discriminate among true and near cognate substrates. To date, analysis of aaRSs function, including identification of residues of aaRS participating in amino acid and tRNA discrimination, has largely relied on the steady state kinetic pyrophosphate exchange and aminoacylation assays. Pre-steady state kinetic studies investigating a more limited set of aaRS systems have also been undertaken to assess the energetic contributions of individual enzyme-substrate interactions, particularly in the adenylation half reaction. More recently, a renewed interest in the use of rapid kinetics approaches for aaRSs has led to their application to several new aaRS systems, resulting in the identification of mechanistic differences that distinguish the two structurally distinct aaRS classes. Here, we review the techniques for thermodynamic and kinetic analysis of aaRS function. Following a brief survey of methods for the preparation of materials and for steady state kinetic analysis, this review will describe pre-steady state kinetic methods employing rapid quench and stopped-flow fluorescence for analysis of the activation and aminoacyl transfer reactions. Application of these methods to any aaRS system allows the investigator to derive detailed kinetic mechanisms for the activation and aminoacyl transfer reactions, permitting issues of substrate specificity, stereochemical mechanism, and inhibitor interaction to be addressed in a rigorous and quantitative fashion.

show abstract

“…Aminoacylation assays were adapted from a recently described procedure (30). Aminoacylation reactions (10 l) were carried out at 37°C (unless otherwise noted) in 100 mM Na-Hepes (pH 7.2)/25 mM MgCl 2 /60 mM NaCl/5 mM ATP/1 mM DTT/10 mM Cyc/tRNA 3Ј-labeled with [␣-32 P]ATP ranging in concentration from 0.25 to 5 times K M concentrations.…”

Section: Purification Of Recombinant M Barkeri Pylrs and In Vitro Trmentioning

confidence: 99%

Pyrrolysine is not hardwired for cotranslational insertion at UAG codons

Ambrogelly¹,

Gundllapalli²,

Herring³

et al. 2007

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

119

126

View full text Add to dashboard Cite

Pyrrolysine (Pyl), the 22nd naturally encoded amino acid, gets acylated to its distinctive UAG suppressor tRNA Pyl by the cognate pyrrolysyl-tRNA synthetase (PylRS). Here we determine the RNA elements required for recognition and aminoacylation of tRNA Pyl in vivo by using the Pyl analog N--cyclopentyloxycarbonyl-L-lysine. Forty-two Methanosarcina barkeri tRNA Pyl variants were tested in Escherichia coli for suppression of the lac amber A24 mutation; then relevant tRNA Pyl mutants were selected to determine in vivo binding to M. barkeri PylRS in a yeast three-hybrid system and to measure in vitro tRNA Pyl aminoacylation. tRNA Pyl identity elements include the discriminator base, the first base pair of the acceptor stem, the T-stem base pair G51:C63, and the anticodon flanking nucleotides U33 and A37. Transplantation of the tRNA Pyl identity elements into the mitochondrial bovine tRNA Ser scaffold yielded chimeric tRNAs active both in vitro and in vivo. Because the anticodon is not important for PylRS recognition, a tRNA Pyl variant could be constructed that efficiently suppressed the lac opal U4 mutation in E. coli. These data suggest that tRNA Pyl variants may decode numerous codons and that tRNA Pyl :PylRS is a fine orthogonal tRNA:synthetase pair that facilitated the late addition of Pyl to the genetic code.orthogonal tRNA ͉ suppression ͉ tRNA identity ͉ pyrrolysyl-tRNA synthetase ͉ aminoacyl-tRNA synthetase

show abstract

A new assay for tRNA aminoacylation kinetics

Cited by 56 publications

References 24 publications

Modulation of tRNA ^Ala identity by inorganic pyrophosphatase

Modulation of tRNA ^Ala identity by inorganic pyrophosphatase

Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases

Pyrrolysine is not hardwired for cotranslational insertion at UAG codons

Contact Info

Product

Resources

About

A new assay for tRNA aminoacylation kinetics

Cited by 56 publications

References 24 publications

Modulation of tRNA Ala identity by inorganic pyrophosphatase

Modulation of tRNA Ala identity by inorganic pyrophosphatase

Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases

Pyrrolysine is not hardwired for cotranslational insertion at UAG codons

Contact Info

Product

Resources

About

Modulation of tRNA ^Ala identity by inorganic pyrophosphatase

Modulation of tRNA ^Ala identity by inorganic pyrophosphatase