An intersubunit contact stimulating transcription initiation byE. coliRNA polymerase: interaction of the α C-terminal domain and ς region 4

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Abstract
The C-terminal domain of the Escherichia coli RNA polymerase (RNAP) alpha subunit (αCTD) stimulates transcription initiation by interacting with upstream (UP) element DNA and a variety of transcription activators. Here we identify specific substitutions in region 4.2 of sigma 70 (ς70) and in αCTD that decrease transcription initiation from promoters containing some, but not all, UP elements. This decrease in transcription derives from a decrease in the initial equilibrium constant for RNAP binding (KB). The open complexes formed by the mutant and wild-type RNAPs differ in DNAse I sensitivity at the junction of the αCTD and ς DNA binding sites, correlating with the differences in transcription. A model of the DNA–αCTD–ς region 4.2 ternary complex, constructed from the previously determined X-ray structures of the Thermus aquaticusς region 4.2–DNA complex and the E. coli αCTD–DNA complex, indicates that the residues identified by mutation in ς region 4.2 and in αCTD are in very close proximity. Our results strongly suggest that αCTD, when bound to an UP element proximal subsite, contacts the RNAP ς70 subunit, increasing transcription. Previous data from the literature suggest that this same ς–αCTD interaction also plays a role in transcription factor-mediated activation.

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