A domain in the N-terminal extension of class IIb eukaryotic aminoacyl-tRNA synthetases is important for tRNA binding

Abstract

Cytoplasmic aspartyl-tRNA synthetase (AspRS) from Saccharomyces cerevisiae is a homodimer of 64 kDa subunits. Previous studies have emphasized the high sensitivity of the N-terminal region to proteolytic cleavage, leading to truncated species that have lost the first 20-70 residues but that retain enzymatic activity and dimeric structure. In this work, we demonstrate that the N-terminal extension in yeast AspRS participates in tRNA binding and we generalize this finding to eukaryotic class IIb aminoacyl-tRNA synthetases. By gel retardation studies and footprinting experiments on yeast tRNA(Asp), we show that the extension, connected to the anticodon-binding module of the synthetase, contacts tRNA on the minor groove side of its anticodon stem. Sequence comparison of eukaryotic class IIb synthetases identifies a lysine-rich 11 residue sequence ((29)LSKKALKKLQK(39) in yeast AspRS with the consensus xSKxxLKKxxK in class IIb synthetases) that is important for this binding. Direct proof of the role of this sequence comes from a mutagenesis analysis and from binding studies using the isolated peptide.

A mechanism for functional segregation of mitochondrial and cytosolic genetic codes

“…Additional domains that confer tRNA affinity are common in tRNA synthetases. These structures typically play no role in the aminoacylation reaction but provide the enzyme with additional binding energy or improve tRNA discrimination (18).…”

Arc1p Organizes the Yeast Aminoacyl-tRNA Synthetase Complex and Stabilizes Its Interaction with the Cognate tRNAs

“…Fusion of this sequence to the E. coli glutaminyl-tRNA synthetase, which lacks a similar domain, renders the bacterial enzyme capable of substituting yeast glutaminyl-tRNA synthetase both in vitro and in vivo (13). Yeast aspartyl-tRNA synthetase also contains an N-terminal extension that can bind to tRNA (16). This domain is not found in the prokaryotic homologue but is conserved in other eukaryotic class IIb synthetases.…”

Aminoacyl-tRNA synthetase complexes: beyond translation

“…Interestingly, these motifs are also involved in protein-protein interactions (Rho et al, 1996;Rho et al, 1998). DRS and KRS also contain helical tRNA-binding domains (TRBD; blue boxes), although they are not related to the motif mentioned above (Frugier et al, 2000;Francin et al, 2002). By contrast, the oligonucleotidebinding (OB) fold domains (blue boxes) in p43 and YRS are related (Renault et al, 2001).…”

“…Interestingly, many ARSs also have cis-acting domains that appear to facilitate the recruitment of tRNAs to their catalytic sites (Cahuzac et al, 2000;Frugier et al, 2000;Francin et al, 2002;Francin and Mirande, 2003). For example, the C-terminus of E. coli MRS (Morales et al, 1999), and the N-terminus of the E. coli FRS β-subunit (Simos et al, 1996) (Fig.…”

“…However, we cannot exclude the alternative possibility -that they were inserted into the ARS structure to augment the catalytic efficiency of the enzymes. Some ARSs also contain cis-acting motifs that are structurally unrelated to the common domains found in Arc1p, p43 and Trbp111 (Cahuzac et al, 2000;Frugier et al, 2000;Kaminska et al, 2001;Francin et al, 2002;Francin and Mirande, 2003) (Figs 2, 4). They usually form amphiphatic helices in which one side of the helix displays an array of basic residues for interaction with tRNAs (Fig.…”