Cloning and expression of the gene encoding RNA polymerase alpha subunit from alkaliphilic Bacillus sp. strain C-125.
The rpoA gene, encoding the alpha subunit of RNA polymerase, was isolated from alkaliphilic Bacillus sp. strain C-125 by the PCR method. A 3-kb HindIII fragment containing the complete rpoA gene was cloned and sequenced. The alpha subunit gene was found to encode a protein consisting of 314 amino acid residues with a molecular mass of 34,805 Da. Compared with the amino acid sequences of other known eubacterial RNA polymerase alpha subunits, the gene has 84% identity to that of B. subtilis, while showing 48% and 47% identity to that of Streptomyces coelicolor and Escherichia coli, respectively. Six conserved regions, which are observed in the case of other eubacteria, were found in the RNA polymerase alpha subunit of this strain. Five of them are located in the N-terminal domain involved in assembly of the core enzyme, while one is located in the C-terminal domain, which interacts with several transcriptional factors and a specific DNA element. By means of recombinant plasmids, a hexahistidine-tagged derivative of the RNA polymerase alpha subunit of strain C-125 and two deletion derivatives (C- and N-terminal domains) of this protein were overexpressed in E. coli cells and purified to near homogeneity.
Nakasone
K
K
DEEP STAR Group, Japan Marine Science and Technology Center, Kanagawa, Japan. nakasone@jamstec.go.jp
Takaki
Y
Y
Takami
H
H
Inoue
A
A
Horikoshi
K
K
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
D012321:DNA-Directed RNA Polymerases; C113275:RNA polymerase alpha subunit