title: 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.
authors.affiliation: School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia.
authors.forename: Xiao
authors.initials: X
authors.lastname: Yang
authors.affiliation: School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia.
authors.forename: Cong
authors.initials: C
authors.lastname: Ma
authors.affiliation: School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia. Electronic address: Peter.Lewis@newcastle.edu.au.
authors.forename: Peter
authors.initials: P
authors.lastname: Lewis
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017931:DNA Primers / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D017931:DNA Primers; D029968:Escherichia coli Proteins; C540081:rpoB protein, E coli; C108570:beta' subunit of RNA polymerase; D012321:DNA-Directed RNA Polymerases
keywords: Dual-expression; Escherichia coli; RNA polymerase; Transcription