Molecular cloning, sequence, structural analysis and expression of the histidyl-tRNA synthetase gene from Streptococcus equisimilis

Corazon A. Menguito, Michael J. Keherly, Chuan Ye Tang, John Papaconstantinou, Paul H. Weigel

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

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 (∼ 58% and ∼ 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 (Mr = 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 tRNAHis in vitro.

Original languageEnglish (US)
Pages (from-to)615-620
Number of pages6
JournalNucleic Acids Research
Volume21
Issue number3
StatePublished - 1993

Fingerprint

Histidine-tRNA Ligase
Cloning
Molecular Cloning
Structural Analysis
Streptococcus
Structural analysis
Sequence Analysis
Genes
Gene
Proteins
Protein
Amino acids
Amino Acids
Fusion
Fusion reactions
Promoter
Escherichia Coli
Nucleotides
Escherichia coli
RNA, Transfer, His

ASJC Scopus subject areas

  • Genetics
  • Statistics, Probability and Uncertainty
  • Applied Mathematics
  • Health, Toxicology and Mutagenesis
  • Toxicology
  • Genetics(clinical)

Cite this

Molecular cloning, sequence, structural analysis and expression of the histidyl-tRNA synthetase gene from Streptococcus equisimilis. / Menguito, Corazon A.; Keherly, Michael J.; Tang, Chuan Ye; Papaconstantinou, John; Weigel, Paul H.

In: Nucleic Acids Research, Vol. 21, No. 3, 1993, p. 615-620.

Research output: Contribution to journalArticle

Menguito, Corazon A. ; Keherly, Michael J. ; Tang, Chuan Ye ; Papaconstantinou, John ; Weigel, Paul H. / Molecular cloning, sequence, structural analysis and expression of the histidyl-tRNA synthetase gene from Streptococcus equisimilis. In: Nucleic Acids Research. 1993 ; Vol. 21, No. 3. pp. 615-620.
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