Human glycyl-tRNA synthetase. Wide divergence of primary structure from bacterial counterpart and species-specific aminoacylation.

K Shiba,P Schimmel,H Motegi,T Noda

doi:10.1016/s0021-9258(18)43986-5

Abstract

Several class I and class II human tRNA synthetases are clearly related to their bacterial counterparts. We report here the cloning, cDNA sequence, deduced primary structure, and expression in bacteria of a class II human glycyl-tRNA synthetase. While the human sequence aligns well with a Bombyx mori and a Saccharomyces cerevisiae sequence for glycyl-tRNA synthetase, particularly in the region of the class II-defining sequence motifs, it diverges widely from that of the Escherichia coli enzyme. The divergence is so great that from the sequences alone we cannot conclude that the human and E. coli proteins are descended from homologous genes. Moreover, even though the human and E. coli class II alanyl-tRNA synthetases cross-acylate their respective tRNAs, aminoacylations by the recombinant human and E. coli glycyl-tRNA synthetases are restricted to their homologous tRNAs. The species-specific aminoacylations correlate with a nucleotide sequence difference at a location in the acceptor stem that is known to be critical for aminoacylations by the E. coli enzyme. Thus, glycyl-tRNA synthetase may have followed a path of historical development different in at least some respects from that of several other tRNA synthetases.

Highlights

Several classI and class I1 human tRNA synthetases operational RNA code for amino acids that possibly pre-dates are clearly related to their bacterial counterparts
Thespecies-speond domain of the classI Escherichia coli methionyl-tRNA synthetase, which is located in the C-terminal haloff the protein, has a predominantly a-helical secondary structure (91, as do the closely related cysteinyl, leucyl, isoleucyl, and valyltRNA syntheses [15,16,17], while the C-terminal domain of the E. coli class I glutaminyl-tRNA synthetase is predominantly cificaminoacylationscorrelatewithanucleotide se- composed of p-structure [10].This second, idiosyncratic protein quence difference at a location in the acceptor stemdotmhaaitn provides for interactions with the seconddomain of tRNAs and, in contrast to the classenzyme.,glycyl-tRNAsynthetasemayhavefoldefining domain, is far lecsosnserved through evolution for the lowed a path of historical development different in at same synthetase
Motif N comprises about 37 amino acids located near the N terminus of several eukaryote tRNA synthetases, including B. mori glycyl-tRNA

Summary

WIDE DIVERGENCE OF PRIMARY STRUCTURE FROM BACTERIAL COUNTERPART AND

Insertions into and flanking tchleass- 303 amino acids, respectively, and thecoding sequences for the defining catalyticcore provide for interactions with theaccep- two subunits are transcribefdrom a single promoter toyield an tor helix domains of tFtNAs, so that a limited part of at least mRNA that has the a-chain coding sequence followed by that some synthetases may be sufficient to aminoacylate RNA oli- for the p-chain, with a n intervening internal translational stop gonucleotides based on the acceptor stems of the cognate codon (UAA “ocher” codon) and a re-initiation signal [23]. Unlike the classI1 alanine enzymes, whose class 11-defining domain is well conserved between E. coli and B. mori [26], the primary sequence of the 687 amino acid B. mori glycyl-tRNA synthetase polypeptide has been shown by Nada et al [27] to possess no similarity to the sequenceof either subunit of the E. coli enzyme [27].

TABLEI Strategy for design of oligonucleotide primers

GTRAAYTCIAKIACICKIAGYADICC GTRAAYTCIAKIACICKIARRAGICC

EXPERIMENTAL PROCEDURES

TVF CIL SVF

RESULTS

DISCUSSION