Abstract
Recent determinations of the structures of the bacterial RNA polymerase (RNAP) and promoter complex thereof establish that RNAP functions as a complex molecular machine that contains distinct structural modules that undergo major conformational changes during transcription. However, the contribution of the RNAP structural modules to transcription remains poorly understood. The bacterial core RNAP (alpha(2)beta beta'omega; E) associates with a sigma (sigma) subunit to form the holoenzyme (E sigma). A mutation removing the beta subunit flap domain renders the Escherichia coli sigma(70) RNAP holoenzyme unable to recognize promoters. sigma(54) is the major variant sigma subunit that utilizes enhancer-dependent promoters. Here, we determined the effects of beta flap removal on sigma(54)-dependent transcription. Our analysis shows that the role of the beta flap in sigma(54)-dependent and sigma(70)-dependent transcription is different. Removal of the beta flap does not prevent the recognition of sigma(54)-dependent promoters, but causes multiple defects in sigma(54)-dependent transcription. Most importantly, the beta flap appears to orchestrate the proper formation of the E sigma(54) regulatory center at the start site proximal promoter element where activator binds and DNA melting originates.
Highlights
Multisubunit DNA-dependent RNA polymerases (RNAP)1 are complex molecular machines that synthesize a RNA copy from a DNA template
RNAP holoenzymes and undergo conformational changes, which orientate and position 70 DNA-binding domains within the RNAP holoenzyme to allow promoter recognition (2, 3). The importance of these conformational changes is underlined by our recent observation that removal of the E. coli RNAP  flap domain abolished the ability of the mutant E70 to recognize promoters of the Ϫ10/Ϫ35 class (13)
A fixed amount of 32P-labeled 54 (32P-54) was combined with various amounts of ⌬flapE or control wild-type core RNAP and the mixtures were separated on a native polyacrylamide gel (Fig. 2a, (i)) and quantified using a PhosphorImager (Fig. 2a (ii)) (10, 20)
Summary
Multisubunit DNA-dependent RNA polymerases (RNAP) are complex molecular machines that synthesize a RNA copy from a DNA template. RNAP holoenzymes that recognize promoters and form transcriptionally competent promoter complexes in the absence of other factors or energy sources. This family, which includes most bacterial factors is named after the prototypical housekeeping of E. coli, 70. RNAP holoenzymes and undergo conformational changes, which orientate and position 70 DNA-binding domains within the RNAP holoenzyme to allow promoter recognition (2, 3) The importance of these conformational changes is underlined by our recent observation that removal of the E. coli RNAP  flap domain abolished the ability of the mutant E70 to recognize promoters of the Ϫ10/Ϫ35 class (13). We studied the properties of E. coli E54 reconstituted from mutant core RNAP harboring the  flap deletion, ⌬885–914 (hereafter called ⌬flapE), to gain insights into the contribution of a core RNAP structural module, which is critical for transcription initiation by E70, to enhancer-dependent transcription by E54
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