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A domain in the N‐terminal extension of class IIb eukaryotic aminoacyl‐tRNA synthetases is important for tRNA binding
Author(s) -
Frugier Magali,
Moulinier Luc,
Giegé Richard
Publication year - 2000
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/19.10.2371
Subject(s) - biology , transfer rna , amino acyl trna synthetases , aminoacyl trna synthetase , binding domain , terminal (telecommunication) , genetics , binding site , biochemistry , rna , gene , telecommunications , computer science
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.