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

摘要

We luivc cloned the gene encoding RNA polymerase a subunit from a gene library of deep-sea barophilic bacterium strain DB6705. The clone conUiins the genes for ribosomal protein S4, RNA polymerase subunit a. and ribosomal protein L17 in this order. The a gene has 32K amino acids and a molecular mass of 36 100 Da with 86.9% identity to Escherichia coli a gene. Differences between the two Ncquunccs were mainly in the N-terminal portion of the u subunit, which is involved in the assembly of the core RNA polymerase; while the K7 C-tenninal residues, which form a region involved in contact with some positive regulators and rrnB PI promoter region called UP-elemuiK, were identical in the both strain. Plasnn'd encoding the a subunit with an N-terminal hexahistidine tag was constructed. Using the plasmid, the reeombinant fusion a subunit was overcxpressed and successfully purified to near homogeneity.