Open AccessUsing Disulfide Bond Engineering To Study Conformational Changes in the β′260-309 Coiled-Coil Region of Escherichia coli RNA Polymerase during σ 70 Binding
Author(s) -
Larry C. Anthony,
Alan A. Dombkowski,
Richard R. Burgess
Publication year - 2002
Publication title -
journal of bacteriology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.652
H-Index - 246
eISSN - 1067-8832
pISSN - 0021-9193
DOI - 10.1128/jb.184.10.2634-2641.2002
Subject(s) - coiled coil , biophysics , rna polymerase , biology , polymerase , conformational change , escherichia coli , dna , biochemistry , transcription (linguistics) , protein subunit , t7 rna polymerase , gene , linguistics , philosophy , bacteriophage
RNA polymerase of Escherichia coli is the sole enzyme responsible for mRNA synthesis in the cell. Upon binding of a sigma factor, the holoenzyme can direct transcription from specific promoter sequences. We have previously defined a region of the beta' subunit (beta'260-309, amino acids 260 to 309) which adopts a coiled-coil conformation shown to interact with sigma(70) both in vitro and in vivo. However, it was not known if the coiled-coil conformation was maintained upon binding to sigma(70). In this work, we engineered a disulfide bond within beta'240-309 that locks the beta' coiled-coil region in the coiled-coil conformation, and we show that this "locked" peptide is able to bind to sigma(70). We also show that the locked coiled-coil is capable of inducing a conformational change within sigma(70) that allows recognition of the -10 nontemplate strand of DNA. This suggests that the coiled-coil does not adopt a new conformation upon binding sigma(70) or upon recognition of the -10 nontemplate strand of DNA.