Pyrophosphate‐caused inhibition of the aminoacylation of tRNA by the leucyl‐tRNA synthetase from <i>Neurospora crassa</i>

Rk, Airas; Cramer, Friedrich

doi:10.1111/j.1432-1033.1986.tb09970.x

Cited by 15 publications

(11 citation statements)

References 30 publications

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“…PPase is an essential protein, and experiments regulating its intracellular concentration reveal that cellular growth is inhibited when PP i concentrations exceed 2-3 mM (43). Surprisingly, typical PP i concentrations in log-phase E. coli cells are between 0.5 and 1 mM (43,44), well above the concentration at which it fully inhibits tRNA synthetases in vitro (21,45). Perhaps the majority of the PP i is sequestered in an intracellular compartment such that it is unavailable to inhibit aaRSs.…”

Section: Discussionmentioning

confidence: 99%

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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Section: Discussionmentioning

confidence: 99%

Modulation of tRNA ^Ala identity by inorganic pyrophosphatase

Wolfson

Uhlenbeck

2002

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

124

163

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“…The rate equation (Eqn 3) can be derived for the mechanism using ordinary principles of writing rate equations (as also in [9]). Fig.…”

Section: Discussionmentioning

confidence: 99%

“…To keep the free Mg2+ concentration constant at 1 mM, 1 shows an assay of the dissociation constants of isoleucine and ATP from the isoleucyl-tRNA synthetase using the intersection points [9] Further data concerning the curvature of the Hanes plots are presented in Fig. 2.…”

Section: Fig 3 Rate Of Uminoacylation As a Function Of Atp Concentrmentioning

confidence: 99%

ATP‐induced activation of the aminoacylation of tRNA by the isoleucyl‐tRNA synthetase from Escherichia coli

Airas

1988

European Journal of Biochemistry

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The rate of aminoacylation of tRNA catalyzed by the isoleucyl-tRNA synthetase form Escherichia coli has been measured. A steady-state kinetic analysis of the rate as a function of the concentration of ATP gave nonlinear Hanes plots. ATP behaves as an activator of the reaction. The activation is observed at a low magnesium ion concentration and in the presence of spermidine. The presence of inorganic pyrophosphate or AMP enhances the activation. The results are consistent with a mechanism in which the binding of a second molecule of ATP increases the rate of dissociation of Ile-tRNA from the enyzme.ATP, together with isoleucine and tRNA, is a normal substrate of the isoleucyl-tRNA synthetase: normal Michaelis-Menten kinetics for the formation of isoleucyl-tRNA has been observed over a wide range of ATP concentrations [1, 21. However, there are also some observations of a more complicated role of ATP in the reaction. In a careful steadystate kinetic analysis of the reaction mechanism of the isoleucyl-tRNA synthetase from Escherichia coli, Freist et al.[ 11 suggest an almost random binding of all three substrates and thereafter an apparently random release of the products. However, after the formation of the enzyme-isoleucyladenylate complex, a subsequent binding of a second molecule of ATP was postulated, in order to account for the inhibition patterns by some ATP analogs. Despite the involvement of two molecules of ATP, the double-reciprocal plots were linear (ATP as the variable substrate).Another note about abnormal ATP kinetics by McNeil and Schimmel [3] showed that ATP inhibits the ATP-PPi isotope exchange reaction. It was suggested that ATP could bind to the PPi site after formation of the isoleucyl-adenylate.In the present study we found conditions of low Mg2+ concentration plus spermidine, where the kinetics of dependence of rate on ATP concentration appeared to show activation by ATP. This anomaly is also consistent with a mechanism in which a second ATP molecule is bound to the enzyme, either to a second binding site or to the normal binding site after formation of isoleucyl-adenylate. MATERIALS AND METHODSThe isoleucyl-tRNA synthetase from E. coli

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“…The effect of PP, on the aminoacyl-tRNA synthetase reaction has already been considered in two earlier papers on this work [7, 81. The present work uses the kinetic equations derived in the preceding paper [9].…”

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confidence: 98%

Effect of inorganic pyrophosphate on the pretransfer proofreading in the isoleucyl‐tRNA synthetase from Escherichia coli

Airas

1992

European Journal of Biochemistry

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A total rate equation was used to calculate the discrimination of valine by the isoleucyl-tRNA synthetase from Escherichia coli. The PPi present in the cell makes the backward reaction or the pyrophosphorolysis of the E .possible. If the E . Ile-AMP has been corrected for wrong aminoacyl adenylation by the pretransfer proofreading, the pyrophosphorolysis rapidly equilibrates the corrected E . Ile-AMp with E . and thus spoils the effect of the proofreading. The loss of the corrected species is avoided if there is a barrier (perhaps conformational) formed by a slow reaction step between the noncorrected E . l'e-AMP and the corrected *E$&Mp. If such a slow conformational change exists, the increase in accuracy from the pretransfer proofreading would be benefitial, and, in addition, the PPi increases the accuracy by optimizing the initial discrimination of the wrong amino acid.The activation of amino acids by the aminoacyl-tRNA synthetases produces PP,, which is able to bind to the aminoacyl adenylate in the reversc reaction [I]. Both the activation and the pyrophosphorolysis are rapid reactions [2] which allow an equilibrium at this step in the presence of PPi. The PPi strongly shifts the equilibrium from the aminoacyl adenylate intermediates towards the start of the total reaction. PP, is decomposed by the inorganic pyrophosphatase present in all living cells. The pyrophosphatase, however, does not destroy all PP,, but a moderate concentration is maintained. The PP, concentration in Escherichia roli cells, thoroughly studied in this laboratory [3 -51, is about 0.5 mM and is not changed very much by varying the conditions.The amino acid specificity of the aminoacyl-tRNA synthetases has been described to be increased in four steps [6]. Two initial discrimination steps occur before the activation reaction, the pretransfer proofreading works at the aminoacyl adenylate step and the posttransfer proofreading at the aminoacyl-tRNA step (Eqn 1).The effect of PP, on the aminoacyl-tRNA synthetase reaction has already been considered in two earlier papers on this work [7, 81. The present work uses the kinetic equations derived in the preceding paper [9]. The roles of PP, in the initial discriminations and pretransfer proofreading were calculated. MATERIALS AND METHODS EquationsThe rate equations were derived as suggested by Cha [lo] and as described in detail in the preceding paper [9]. The reaction scheme was divided in separate rapid equilibrium segments. Since the amino acid specificity is to be examined, the binding reaction for the amino acid makes one boundary of the segments, and the different amino acids, isoleucine and valine, have their own segments (Eqn 2).

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Pyrophosphate‐caused inhibition of the aminoacylation of tRNA by the leucyl‐tRNA synthetase from Neurospora crassa

Cited by 15 publications

References 30 publications

Modulation of tRNA ^Ala identity by inorganic pyrophosphatase

Modulation of tRNA ^Ala identity by inorganic pyrophosphatase

ATP‐induced activation of the aminoacylation of tRNA by the isoleucyl‐tRNA synthetase from Escherichia coli

Effect of inorganic pyrophosphate on the pretransfer proofreading in the isoleucyl‐tRNA synthetase from Escherichia coli

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Pyrophosphate‐caused inhibition of the aminoacylation of tRNA by the leucyl‐tRNA synthetase from Neurospora crassa

Cited by 15 publications

References 30 publications

Modulation of tRNA Ala identity by inorganic pyrophosphatase

Modulation of tRNA Ala identity by inorganic pyrophosphatase

ATP‐induced activation of the aminoacylation of tRNA by the isoleucyl‐tRNA synthetase from Escherichia coli

Effect of inorganic pyrophosphate on the pretransfer proofreading in the isoleucyl‐tRNA synthetase from Escherichia coli

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Modulation of tRNA ^Ala identity by inorganic pyrophosphatase

Modulation of tRNA ^Ala identity by inorganic pyrophosphatase