Cysteinyl-tRNA synthetase from Saccharomyces cerevisiae. Purification, characterization and assignment to the genomic sequence YNL247w.
Cysteinyl-tRNA synthetase (CRS) from Saccharomyces cerevisiae was purified 2300-fold with a yield of 33%, to a high specific activity (kcat4.3 s-1 at 25 degrees C for the aminoacylation of yeast tRNACys). SDS-PAGE revealed a single polypeptide corresponding to a molecular mass of 86 kDa. Polyclonal antibodies to the purified protein inactivated CRS activity and detected only one polypeptide of 86 kDa in a yeast extract subjected to SDS-PAGE followed by immunoblotting. In contrast to bacterial CRS which is a monomer of about 50 kDa, the native yeast enzyme behaved as a dimer, as assessed by gel filtration and cross-linking. Its subunit molecular mass is in good agreement with the value of 87.5 kDa calculated for the protein encoded by the yeast genomic sequence YNL247w. The latter was previously tentatively assigned to CRS, based on limited sequence similarities to the corresponding enzyme from other sources. Determination of the amino acid sequence of internal polypeptides derived from the purified yeast enzyme confirmed this assignment. Alignment of the primary sequences of prokaryotic and yeast CRS reveals that the larger size of the latter is accounted for mostly by several insertions within the sequence.
Motorin
Y
Y
Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, Gif-sur-Yvette, France.
Le Caer
J P
JP
Waller
J P
JP
D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D000946:Antigens, Fungal / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016681:Genome, Fungal; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D017421:Sequence Analysis
D000946:Antigens, Fungal; D005656:Fungal Proteins; D000604:Amino Acyl-tRNA Synthetases; C019975:cysteinyl-tRNA synthetase