Pathway of promoter melting by Bacillus subtilis RNA polymerase at a stable RNA promoter: Effects of temperature, .delta. protein, and .sigma. factor mutations

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Abstract
Bacillus subtilis RNA polymerase (RNAP) contains a catalytic core (beta beta'alpha(2); or E) associated with one of several sigma factors, which determine promoter recognition, and delta protein, which enhances promoter selectivity. We have shown previously that specific mutations in sigma(A) region 2.3, or addition of delta, decrease the ability of RNAP to melt the ilv-leu promoter. Here we extend these studies to a stable RNA promoter, P-tmS, which controls transcription of seven tRNA genes. KMnO4 footprinting was used to visualize DNA melting at P-tmS as a function of both temperature and the protein composition of the RNAP holoenzyme. We propose that the pathway leading to productive initiation includes several intermediates: a closed complex (RP(c)), a complex in which DNA melting has nucleated within the conserved TATA element (RP(n)), and an open complex in which DNA-melting extends to at least -4 (RP(o1)). RNAP reconstituted with either of two mutant sigma(A) proteins, Y189A and W192A, was defective for both the nucleation and propagation of the transcription bubble while a third sigma(A) mutant, W193A, allows normal nucleation of DNA-melting, but does not efficiently propagate the melted region downstream.