title: A novel method for the production of in vivo-assembled, recombinant Escherichia coli RNA polymerase lacking the α C-terminal domain.
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.
authors.affiliation: The Rockefeller University, Laboratory of Molecular Biophysics, 1230 York Avenue, New York, New York 10065, USA.
authors.forename: Kelly-Anne
authors.initials: KA
authors.lastname: Twist
authors.forename: Seyyed I
authors.initials: SI
authors.lastname: Husnain
authors.forename: Josef D
authors.initials: JD
authors.lastname: Franke
authors.forename: Deepti
authors.initials: D
authors.lastname: Jain
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Campbell
authors.forename: Bryce E
authors.initials: BE
authors.lastname: Nickels
authors.forename: Mark S
authors.initials: MS
authors.lastname: Thomas
authors.forename: Seth A
authors.initials: SA
authors.lastname: Darst
authors.forename: Lars F
authors.initials: LF
authors.lastname: Westblade
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D017384:Sequence Deletion; D015854:Up-Regulation
chemical_list: D021122:Protein Subunits; D011994:Recombinant Proteins; D012321:DNA-Directed RNA Polymerases