Promoter opening by ς54 and ς70 RNA polymerases: ς factor-directed alterations in the mechanism and tightness of control

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Abstract
Transcription control at the melting step is not yet understood. Here, band shift, cross-linking, and transcription experiments on diverse DNA probes were used with two bacterial RNA polymerase holoenzymes that differ in how they regulate melting. Data indicated that both ς54 and ς70 holoenzymes assume a default closed form that cannot establish single-strand binding. Upon activation the enzymes are converted to an open form that can bind simultaneously to the upstream fork junction and to the melted transcription start site. The key difference is that ς54imposes tighter regulation by creating a complex molecular switch at −12/−11; the current data show that this switch can be thrown by activator. In this case an ATP-bound enhancer protein causes ς54 to alter its cross-linking pattern near −11 and also causes a reorganization of holoenzyme: DNA interactions, detected by electrophoretic mobility-shift assay. At a temperature-dependent ς70 promoter, elevated temperature alone can assist in triggering conformational changes that enhance the engagement of single-strand DNA. Thus, the two ς factors modify the same intrinsic opening pathway to create quite different mechanisms of transcriptional regulation.