Modulation of tRNA ^Ala identity by inorganic pyrophosphatase

Abstract

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...

Structure-specific tRNA Determinants for Editing a Mischarged Amino Acid

“…Mischarging with wildtype AlaRS (5 M) was done in the presence of tRNA Ala transcript (5 M) or minihelix Ala (15 M) and [ 3 H]Gly (10.7 M) in charging buffer at 37°C. Aminoacylation of 32 P-labeled minihelix Ala was followed by the assay described by Wolfson et al (25). The aminoacylation reaction was carried out with 3 mM alanine (or 175 mM glycine), 500 nM AlaRS, and 5.1 M labeled minihelix Ala .…”

“…Mischarged tRNA Ala was produced in vitro using C666A/ Q584H AlaRS, as described previously (19) in aminoacylation buffer (50 mM HEPES, pH 7.5, 20 mM KCl, 0.1 mg/ml bovine serum albumin, 20 mM ␤-mercaptoethanol, and 10 mM MgCl 2 ). Labeling of RNA at the 3Ј-terminal adenosine was accomplished using [␥-32 P]ATP and E. coli tRNA-terminal nucleotidyl transferase, as described by Wolfson et al (25).…”

A continuous assay for monitoring the synthetic and proofreading activities of multiple aminoacyl-tRNA synthetases for high-throughput drug discovery

“…Non-cognate aa are poor substrates for aaRSs and can exhibit a rate of activation for formation of the aminoacyl-adenylate intermediate that is 3 to 5 orders of magnitude lower than that of the cognate aa [31]. PPase in the coupled reaction helps to circumvent this problem because hydrolysis of PPi prevents reversal of the aa activation step and has been shown to drive aminoacylation forward for both cognate and non-cognate aa [29,32]. Thus, in this scheme, a drug targeting either of the aaRS's active sites (synthetic or editing) is predicted to decrease the kinetics of PPi synthesis.…”

Photoreactive Bicyclic Amino Acids as Substrates for Mutant Escherichia coli Phenylalanyl-tRNA Synthetases

“…Pyrophosphate (PP i ) exchange and aminoacylation reactions were performed as described (10), with unlabeled benzofuranylalanine included at 2 and 4 mM for the determination of inhibition constants during aminoacylation. The direct attachment of benzofuranylalanine and benzotriazolylalanine to in vitro transcribed tRNA was monitored by direct 32 P labeling of tRNA Phe using E. coli tRNA-terminal nucleotidyltransferase (15) followed by aminoacylation and product visualization as previously described (16).…”

“…PheRS-␣A294G aminoacylates tRNA Phe with benzofuranylalanine. tRNA Phe was labeled as previously described (16). The reaction contained 100 mM Hepes, pH 7.2, 10 mM MgCl 2 , 30 mM KCl, 2 mM ATP, 1 M E. coli tRNA Phe (Roche Applied Science), and a trace (3.5 nCi) of 3Ј 32 P-labeled tRNA Phe , and aminoacylation was performed at 1 M tRNA and 1 nM wild type PheRS (diamonds and squares) or ␣A294G mutant (triangles and circles) with 2 mM Phe (diamonds and triangles) or 0.2 mM benzofuranylalanine (squares and circles).…”