Molecular cloning of the gene encoding RNA polymerase α subunit from deep-sea barophilic bacterium

Abstract

We have cloned the gene encoding RNA polymerase α subunit from a gene library of deep-sea barophilic bacterium strain DBD6705. The clone contains the genes for ribosomal protein S4, RNA polymerase subunit α and ribosomal protein L17 in this order. The α gene has 328 amino acids and a molecular mass of 36 100 Da with 86.9% identity to Escherichia coli α gene. Differences between the two sequences were mainly in the N-terminal portion of the α subunit, which is involved in the assembly of the core RNA polymerase; while the 87 C-terminal residues, which form a region involved in contact with some positive regulators and rrnB P1 promoter region called UP-element, were identical in the both strain. Plasmid encoding the α subunit with an N-terminal hexahistidine tag was constructed. Using the plasmid, the recombinant fusion α subunit was overexpressed and successfully purified to near homogeneity.