Arginine aminoacylation identity is context-dependent and ensured by alternate recognition sets in the anticodon loop of accepting tRNA transcripts.

Abstract

Colresponding authorYeast arginyl-tRNA synthetase recognizes the nonmodified wild-type transcripts derived from both yeast tRNAArg and tRNAASP with equal efficiency. It discriminates its cognate natural substrate, tRNA , from non-cognate tRNAASP by a negative discrimination mechanism whereby a single methyl group acts as an anti-determinant. Considering these facts, recognition elements responsible for specific arginylation in yeast have been searched by studying the in vitro arginylation properties of a series of transcripts derived from yeast tRNAASP, considered as an arginine isoacceptor tRNA. In parallel, experiments on similar tRNAArg transcripts were performed. Unexpectedly, in the tRNAArg context, arginylation is basically linked to the presence of residue C35, whereas in the tRNAASP context, it is deeply related to that of C36 and G37 but is insensitive to the nucleotide at position 35. Each of these nucleotides present in one host, is absent in the other host tRNA. Thus, arginine identity is dependent on two different specific recognition sets according to the tRNA framework investigated.

The RNA sequence context defines the mechanistic routes by which yeast arginyl-tRNA synthetase charges tRNA

“…Accuracy of translation relies on specific aminoacylation of tRNAs by their cognate aminoacyl-tRNA synthetases (aaRS)+ This specificity is governed by molecular signals within tRNAs, including positive elements responsible for specific recognition by cognate synthetases and negative elements hindering recognition by noncognate enzymes+ Recognition sets have been established in a number of tRNAs (e+g+, Giegé et al+, 1993;McClain, 1993aMcClain, , 1993bSaks et al+, 1994) and were shown to be constituted by a limited number of nucleotides and/or structural features+ The concept of specificity calls for a unique combination of those elements for a given aminoacylation system and indeed many experimental data are in line with this view+ However, for the arginine system, we have demonstrated that yeast arginyl-tRNA synthetase (ArgRS) has the unexpected property of recognizing indiscriminately two alternate sets of nucleotides within two host tRNAs (Sissler et al+, 1996)+ Indeed, ArgRS aminoacylates its major tRNA Arg isoacceptor thanks to the presence in its anticodon loop of C35 and, to a lesser extent, of U36 or G36+ This set is designated as [C35U36] Arg + Additionally, yeast ArgRS is also able to interact rather strongly with native tRNA Asp and to mischarge this molecule with low efficiency (Ebel et al+, 1973;Gangloff et al+, 1973;Perret et al+, 1990a)+ Arginylation becomes efficient with noncognate tRNA Asp so far as it is deprived of modified nucleotides (Perret et al+, 1990a;Pütz et al+, 1994)+ For this unmodified substrate, arginylation is deeply related to the presence of residues C36 and G37, but is insensitive to the nature of nt 35+ This second and alternate recognition set is designated as [C36G37] Asp + Furthermore, contacts of yeast ArgRS on in vitro-transcribed tRNA Arg (derived from the major isoacceptor) and tRNA Asp have been established by footprinting with enzymatic and chemical probes (Sissler et al+, 1997)+ They revealed that both transcripts interact with ArgRS along the D-arm side as typical for class I synthetases, and the anticodon loop, the region that contains the identity nucleotides+ However, details of interaction patterns are idiosyncratic and indicate that recognition is governed by the synthetase+ The existence of two alternate identity sets that trigger catalysis by the same synthetase addresses new questions about the mechanisms leading to aminoacylation specificity+ We recall that transplantation of individual arginine identity elements from one tRNA substrate into the other one has negative consequences on arginylation (Sissler et al+, 1996) and suggests nonclassical behaviors of these elements+ Further, the functional properties of a chimeric tRNA Arg with the anticodon loop of tRNA Asp demonstrated that the framework in which the identity elements are embedded contributes to expression of arginine identity (Sissler et al+, 1996)+ This work investigates the mechanism of arginine identity expression and the precise interrelations between the two arginine identity sets within the tRNA Arg or tRNA Asp frameworks+ Functional analysis of a series o...…”

“…Exchange of individual arginine identity nucleotides between tRNA Arg and tRNA Asp transcripts produces dramatic negative effects on their arginylation capacities (Sissler et al+, 1996)+ To understand the duality of arginine identity expression, two chimera were constructed into which the complete recognition set of the other host molecule was transplanted+ In these tRNAs, residual nucleotides from the native recognition set were removed by mutation+ Sequences of variants, 1 together with those of their wild-type counterparts, are displayed in Figure 1+ Comparison of the kinetic data of the four arginine acceptors also informs about the role of the tRNA frameworks+ First, ArgWT and AspC35U36A37, with the same arginine recognition set but not the framework, have a 4+7-fold difference in K m compensated by a 3+5-fold difference in k cat + Larger effects are observed for AspWT and ArgU35C36G37, where both K m and k cat vary by a factor of ;11+…”

“…1 for cooperativity, and R , 1 for anticooperativity (Pütz et al+, 1993)+ Conversion of ArgWT to ArgU35C36G37 was done progressively (Fig+ 3)+ Individual substitutions of C35 and U36 reduce arginylation, especially in ArgU35 (L ϭ 2670) and more moderately in ArgC36 (L ϭ 45)+ These mutations were useful to define C35 as the major arginine recognition element and U36 as a minor element (Sissler et al+, 1996)+ Replacement of A37 by G37 is without noticeable effect (L ϭ 2+5 in ArgG37 )+ Simultaneous mutation of the three anticodon loop positions leads to efficient ArgU35C36G37 (L ϭ 11)+ Comparison of its experimental efficiency with that calculated from single mutant data (Table 1) highlights an anticooperative relationship between the three nucleotides+ Indeed, in the hypothesis of additivity, L calc would be 2+5 ϫ 45 ϫ 2,670 ϭ 3 ϫ 10 5 , a figure much higher than L exp ϭ 11+ The coupling factor R of 3+6 ϫ 10 Ϫ5 quantifies this strong effect+ A simple interpretation would be that the anticooperativity is due to compensating effects brought by the mutations in the [C35U36] Argrecognition set+ A more subtle interpretation has to take into account that the mutations have replaced this set by the alternate [C36G37] Asp -set+ Because footprinting shows proximity of identity nucleotides with ArgRS (Sissler et al+, 1997) and A37 by a coupling factor R ϭ 4+9 ϫ 10 Ϫ5 + This strong anticooperativity can only result from compensatory effects brought by the mutations at the recognition positions because no positive signal has been introduced, except U36 considered as a minor identity element in the tRNA Arg context+ Replacement in tRNA Asp of U35 by C35 does not affect arginylation (L ϭ 12 compared with L ϭ 10 for AspWT )+ Because of the neutral effect of the C35 mutation, it could be anticipated that its introduction in the double mutant AspU36A37 would not improve its arginylation capacity+ However, the resulting triple variant, AspC35U36A37, is a very efficient substrate (L ϭ 0+8)+ The calculated coupling factor R ϭ 1+24 ϫ 10 Ϫ9 reveals here also strong anticooperative effects+ Because AspC35U36A37 is bearing the complete [C35U36] Arg -recognition set, it is reasonable to assume that anticooperativity is mediated by the creation of novel productive interactions with ArgRS+…”

“…tRNAs were obtained by in vitro transcription of synthetic genes+ Genes encoding wild-type and variant species of yeast tRNA Arg III and tRNA Asp , downstream from the T7 RNA polymerase promoter, were constructed and cloned according to Sissler et al+ (1996)+ We note that tRNA Arg III, the major among the five arginine isoacceptors, is represented by 11 of 19 tRNA Arg genes in the yeast genome (Goffeau et al+, 1996;Hani & Feldmann, 1998; G+ Keith & G+ Dirheimer, in prep+)+ Sequences of tRNAs are according to Gangloff et al+ (1971) and Keith and Dirheimer (1980)+ For tRNA Asp , it has been shown that replacement of U1-A72 by G1-C72, more favorable for transcription, is without detrimental consequence on its activity (Perret et al+, 1990a)+ Plasmids containing genes for tRNAs were in vitro transcribed after linearization as described in Perret et al+ (1990b)+ Transcripts were purified on 12% polyacrylamide/urea gels to single nucleotide resolution, electroeluted, and ethanol precipitated+ Concentration of tRNA stock solutions were determined by absorbance measurements at A 260nm +…”

“…Aminoacylation reactions of transcripts derived from tRNA Arg and tRNA Asp have been performed as described in Sissler et al+ (1996)+ Basically, from 0+2 to 4 mM of transcripts were incubated in 50 mM Hepes-NaOH, pH 7+5, 30 mM KCl, 15 mM MgCl 2 , 10 mM ATP, 2+5 mM glutathione, 50 mM 3 H-labeled arginine, and 0+3-900 nM of yeast ArgRS+ Notice that the amino acid concentration in aminoacylation assays is much higher than the K m of arginine, which is 1+5 mM (Gangloff et al+, 1976), so that kinetic artefacts are prevented+…”