CLONING AND EXPRESSION OF THE GENE ENCODING RNA-POLYMERASE ALPHA-SUBUNIT FROM ALKALIPHILIC BACILLUS SP. STRAIN C-125

The rpoA gene, encoding the or 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 seq uenced. The ex subunit gene was found to encode a protein consisting o f 314 amino acid residues with a molecular mass of 34 805 Da. Compared with the amino acid sequences of other known eubacterial RNA polymera se a 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. Sh conserved regions, which are observ ed in the case of other eubacteria, were found in the RNA polymerase c t subunit of this strain. Five of them are located in the N-terminal d omain involved in assembly of the core enzyme, while one is located in the C-terminal domain, which interacts with several transcriptional f actors and a specific DNA element. By means of recombinant plasmids, a hexahistidine-tagged derivative of the RNA polymerase a subunit of st rain 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. (C) 1998 Federation of European Microbiological Societie s. Published by Elsevier Science B.V. All rights reserved.