PMID:11976292
| Citation |
Anthony, LC, Dombkowski, AA and Burgess, RR (2002) Using disulfide bond engineering to study conformational changes in the beta'260-309 coiled-coil region of Escherichia coli RNA polymerase during sigma(70) binding. J. Bacteriol. 184:2634-41 |
|---|---|
| Abstract |
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. |
| Links | |
| Keywords |
DNA-Directed RNA Polymerases/chemistry; DNA-Directed RNA Polymerases/metabolism; Disulfides/chemistry; Electrophoretic Mobility Shift Assay; Escherichia coli Proteins/chemistry; Oligonucleotides/metabolism; Protein Conformation; Protein Engineering; Sigma Factor/metabolism |
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