Molecular cloning, sequence, structural analysis and expression of the histidyl-tRNA synthetase gene from Streptococcus equisimilis.
The histidyl-tRNA synthetase gene (hisS) from Streptococcus equisimilis was cloned and sequenced. The gene for this aminoacyl-tRNA synthetase has an open reading frame of 1278 nucleotides. The deduced amino acid sequence encodes a protein of 426 amino acids with MW = 47,932. The protein is predicted to be soluble with a pl = 5.27. The protein sequence has extensive overall identity/similarity with the Escherichia coli and the yeast histidyl-tRNA synthetases (approximately 58% and approximately 20%, respectively). A putative promoter for gene transcription lies within two hundred nucleotides of the polypeptide start codon. The enzyme was overexpressed, to a level of about 18% of total cellular protein, as a fusion protein (containing an additional 15 amino acids) in E. coli using the pT7 expression system containing the T7 RNA polymerase/promoter (Tabor and Richardson, Proc. Natl. Acad. Sci. U.S.A. 82:1074-1078, 1985). The predicted MW for the hisS gene product is in good agreement with the size of the fusion protein determined by SDS-PAGE (M(r) = 53,700). Amino acid sequencing of the intact fusion protein and proteolytic fragments confirmed the deduced sequence of the synthetase at many positions throughout the protein. The expressed protein catalyzed the specific aminoacylation of tRNA(His) in vitro.
Menguito
C A
CA
Department of Human Biological Chemistry & Genetics, University of Texas Medical Branch, Galveston 77555-0647.
Keherly
M J
MJ
Tang
C
C
Papaconstantinou
J
J
Weigel
P H
PH
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015870:Gene Expression; D006643:Histidine-tRNA Ligase / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017386:Sequence Homology, Amino Acid; D013291:Streptococcus / Q000201:enzymology / Q000235:genetics*
D004269:DNA, Bacterial; D006643:Histidine-tRNA Ligase