PATHWAY OF PROMOTER MELTING BY BACILLUS-SUBTILIS RNA-POLYMERASE AT A STABLE RNA PROMOTER - EFFECTS OF TEMPERATURE, DELTA-PROTEIN, AND SIGMA-FACTOR MUTATIONS

Bacillus subtilis RNA polymerase (RNAP) contains a catalytic core (bet a beta'alpha(2); or E) associated with one of several sigma factors, w hich determine promoter recognition, and delta protein, which enhances promoter selectivity. We have shown previously that specific mutation s in sigma(A) region 2.3, or addition of delta, decrease the ability o f RNAP to melt the ilv-leu promoter. Here we extend these studies to a stable RNA promoter, P-tmS, which controls transcription of seven tRN A 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 ini tiation includes several intermediates: a closed complex (RP(c)), a co mplex in which DNA melting has nucleated within the conserved TATA ele ment (RP(n)), and an open complex in which DNA-melting extends to at l east -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 efficie ntly propagate the melted region downstream.