A vector system that allows simple generation of mutant Escherichia coli RNA polymerase

Abstract

We describe a dual vector-based system for overproduction of recombinant Escherichia coli RNA polymerase (RNAP). A cleavable deca-histidine tag (His10) was incorporated into the C-terminus of the β′ subunit to facilitate protein purification. Unique restriction sites were introduced into the genes encoding the β and β′ subunits (rpoB and rpoC, respectively), facilitating mutation of functionally significant subunit fragments through insertion of modified PCR fragments into the appropriate vector. RNAP with an R275A substitution in the β′ subunit, which is essential for interaction with transcription initiation factor σ, was generated and exhibited reduced activity compared to native recombinant RNAP.