Function of promoter upstream bent DNA of the Clostridium perfringens phospholipase C gene

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The Clostridium perfringens phospholipase C gene (plc) possesses three phased A-tracts upstream of the promoter (-66 to 40). The A-tracts form bent DNA, facilitate the formation of the RNA polymerase (RNAP)-plc promoter complex through extension of the contact region, and activate the transcription in a low temperature-dependent manner. In order to clarify the mechanism of the transcriptional regulation by the A-tracts, it seems important to elucidate specific subunit/domain of RNAP and the A-tracts responsible for the extended contact.We cloned rpoA encoding α subunit of the C. perfringens RNAP. The deduced amino acid sequence was identical to the N-terminal sequence of purified RNAP α subunit. The coding region of rpoA was cloned into an expression vector to overproduce and purify the α subunit (α-WT, 315 aa). N-terminal (α-NTD, 228 aa) and C-terminal (α-CTD, 79 aa) domains of the subunit were also purified in the same manner.The gel retardation assays showed that α-WT and α-CTD bind to a DNA fragment containing three phased A-tracts and plc promoter (3Ap), but that α-NTD does not. From this result, a possibility was raised that α-CTD binds to the A-tracts. We are trying to show the contact region between 3Ap and α-CTD by hydroxyl radical footprinting at the moment.