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

• A dual-expression vector system for E. coli RNAP overproduction was constructed.
• A number of unique restriction sites were introduced enabling the simple generation of mutant E. coli RNAP subunits.
• Wild type and mutant E. coli RNAP were successfully purified.
• Enzyme with an amino acid change in the major σ70 binding site showed reduced initiation in an in-vitro transcription assay.

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.