A truncated aminoacyl–tRNA synthetase modifies RNA

Abstract

Aminoacyl-tRNA synthetases are modular enzymes composed of a central active site domain to which additional functional domains were appended in the course of evolution. Analysis of bacterial genome sequences revealed the presence of many shorter aminoacyl-tRNA synthetase paralogs. Here we report the characterization of a well conserved glutamyl-tRNA synthetase (GluRS) paralog (YadB in Escherichia coli) that is present in the genomes of >40 species of proteobacteria, cyanobacteria, and actinobacteria. The E. coli yadB gene encodes a truncated GluRS that lacks the C-terminal third of the protein and, consequently, the anticodon binding domain. Generation of a yadB disruption showed the gene to be dispensable for E. coli growth in rich and minimal media. Unlike GluRS, the YadB protein was able to activate glutamate in presence of ATP in a tRNA-independent fashion and to transfer glutamate onto tRNA Asp . Neither tRNA Glu nor tRNA Gln were substrates. In contrast to canonical aminoacyl-tRNA, glutamate was not esterified to the 3 -terminal adenosine of tRNA Asp . Instead, it was attached to the 2-amino-5-(4,5-dihydroxy-2-cyclopenten-1-yl) moiety of queuosine, the modified nucleoside occupying the first anticodon position of tRNA Asp . Glutamyl-queuosine, like canonical Glu-tRNA, was hydrolyzed by mild alkaline treatment. Analysis of tRNA isolated under acidic conditions showed that this novel modification is present in normal E. coli tRNA; presumably it previously escaped detection as the standard conditions of tRNA isolation include an alkaline deacylation step that also causes hydrolysis of glutamyl-queuosine. Thus, this aminoacyl-tRNA synthetase fragment contributes to standard nucleotide modification of tRNA.

Aminoacyl-tRNAs: setting the limits of the genetic code

“…2004;Salazar et al 2004). However, glutamate is not esterified to the normal aminoacylation position at the tRNA's 3Ј-terminal adenosine, but is attached to the hypermodified nucleoside queuosine located in the first position of the tRNA Asp anticodon (Salazar et al 2004). Thus, this aatRNA synthetase fragment contributes to nucleotide modification of tRNA, further broadening the range of functions thus far attributed to synthetase paralogs.…”

Bases of biocontrol: Sequence predicts synthesis and mode of action of agrocin 84, the Trojan Horse antibiotic that controls crown gall

“…Conscription of the adenylylation reaction catalyzed by these enzymes is not without precedent. Truncated paralogues of aaRS enzymes are involved in diverse noncanonical functions, including tRNA modifications (27), and biosynthesis of amino acids and other metabolites (28).…”

Turning tRNA upside down: When aminoacylation is not a prerequisite to protein synthesis

“…Recent studies confirmed YadB as a structural analogue of the catalytic core of GluRS that has retained the ability to activate glutamate (11), albeit without the need for tRNA binding that characterizes GluRS (12). Closer examination revealed that YadB does not attach activated glutamate to either tRNA Gln or tRNA Glu (11), but instead to tRNA Asp (9,13). Although the latter report by Dubois et al (13) in this issue of PNAS provided unequivocal evidence for the surprising aminoacylation of tRNA Asp , it left unanswered the more vexing question of what function, if any, this apparent heterologous aminoacylation serves in vivo.…”

“…The surprising answer to the question of YadB's function is provided by the work of Salazar et al (9). In addition to confirming what was reported concerning YadB (11, 13), Salazar et al establish a truly surprising fate for the amino acid in YadB aminoacylation: rather than being transferred to the 3Ј end of the tRNA, the glutamate becomes attached to the hypermodified nucleoside queuosine (Q) at the first anticodon position, leading to the formation of glutamyl-Q (Fig.…”

“…The lability of the glutamyl-Q linkage in neutral and mildly alkaline solutions virtually guarantees that, under tRNA isolation conditions that promote the formation of deacylated tRNA, the Q34 glutamylation will be lost. When tRNA is isolated under mildly acidic conditions, like those used by Salazar et al (9), the modification is preserved. This finding indicates that the whole question of the methodology of tRNA purification ought to be revisited, because current protocols may remove labile but functionally significant modifications.…”