Identification of an amino acid region supporting specific methionyl-tRNA synthetase: tRNA recognition.
Site-directed nuclease digestion and nonsense mutations of the Escherichia coli metG gene were used to produce a series of C-terminal truncated methionyl-tRNA synthetases. Genetic complementation studies and characterization of the truncated enzymes establish that the methionyl-tRNA synthetase polypeptide (676 residues) can be reduced to 547 residues without significant effect on either the activity or the stability of the enzyme. The truncated enzyme (M547) appears to be similar to a previously described fully active monomeric from of 64,000 Mr derived from the native homodimeric methionyl-tRNA synthetase (2 x 76,000 Mr) by limited trypsinolysis in vitro. According to the crystallographic three-dimensional structure at 2.5 A resolution of this trypsin-modified enzyme, the polypeptide backbone folds into two domains. The former, the N-domain, contain a crevice that is believed to bind ATP. The latter, the C-domain, has a 28 C-residue extension (520 to 547), which folds back, toward the N-domain and forms an arm linking the two domains. This study shows that upon progressive shortening of this C-terminal extension, the enzyme thermostability decreases. This observation, combined with the study of several point mutations, allows us to propose that the link made by the C-terminal arm of M547 between its N and C-terminal domains is essential to sustain an active enzyme conformation. Moreover, directing point mutations in the 528-533 region, which overhangs the putative ATP-binding site, demonstrates that this part of the C-terminal arm participates also in the specific complexation of methionyl-tRNA synthetase with its cognate tRNAs.
Mellot
P
P
Laboratoire de Biochimie, UA 240 du CNRS, Palaiseau, France.
Mechulam
Y
Y
Le Corre
D
D
Blanquet
S
S
Fayat
G
G
D000215:Acylation; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006358:Hot Temperature; D008718:Methionine-tRNA Ligase / Q000378:metabolism*; D008958:Models, Molecular; D010455:Peptides / Q000378:metabolism*; D012329:RNA, Bacterial / Q000235:genetics*; D012343:RNA, Transfer / Q000235:genetics*; D013329:Structure-Activity Relationship; D014357:Trypsin
D010455:Peptides; D012329:RNA, Bacterial; D012343:RNA, Transfer; D014357:Trypsin; D000604:Amino Acyl-tRNA Synthetases; D008718:Methionine-tRNA Ligase