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A novel method for the production of in vivo ‐assembled, recombinant Escherichia coli RNA polymerase lacking the α C‐terminal domain
Author(s) -
Twist KellyAnne,
Husnain Seyyed I.,
Franke Josef D.,
Jain Deepti,
Campbell Elizabeth A.,
Nickels Bryce E.,
Thomas Mark S.,
Darst Seth A.,
Westblade Lars F.
Publication year - 2011
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.622
Subject(s) - recombinant dna , rna polymerase , escherichia coli , mutant , polymerase , protein subunit , biology , in vivo , transcription (linguistics) , biochemistry , protein biosynthesis , microbiology and biotechnology , chemistry , gene , genetics , linguistics , philosophy
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.