A novel method for the production of in vivo-assembled, recombinant Escherichia coli RNA polymerase lacking the α C-terminal domain Academic Article uri icon

Overview

MeSH Major

  • DNA-Directed RNA Polymerases
  • Escherichia coli
  • Protein Engineering
  • Recombinant Proteins

abstract

  • The biochemical characterization of the bacterial transcription cycle has been greatly facilitated by the production and characterization of targeted RNA polymerase (RNAP) mutants. Traditionally, RNAP preparations containing mutant subunits have been produced by reconstitution of denatured RNAP subunits, a process that is undesirable for biophysical and structural studies. Although schemes that afford the production of in vivo-assembled, recombinant RNAP containing amino acid substitutions, insertions, or deletions in either the monomeric β or β' subunits have been developed, there is no such system for the production of in vivo-assembled, recombinant RNAP with mutations in the homodimeric α-subunits. Here, we demonstrate a strategy to generate in vivo-assembled, recombinant RNAP preparations free of the α C-terminal domain. Furthermore, we describe a modification of this approach that would permit the purification of in vivo-assembled, recombinant RNAP containing any α-subunit variant, including those variants that are lethal. Finally, we propose that these related approaches can be extended to generate in vivo-assembled, recombinant variants of other protein complexes containing homomultimers for biochemical, biophysical, and structural analyses.

publication date

  • June 2011

Research

keywords

  • Academic Article

Identity

Language

  • eng

PubMed Central ID

  • PMC3104228

Digital Object Identifier (DOI)

  • 10.1002/pro.622

PubMed ID

  • 21416542

Additional Document Info

start page

  • 986

end page

  • 95

volume

  • 20

number

  • 6