Promoter selectivity of the Bacillus subtilis RNA polymeraseσA and σH holoenzymes

The sigma(H) of Bacillus subtilis directs transcription of a large number of early sporulation genes, whereas the principal sigma factor, sigma(A), is essential for the transcription of the genes for vegetative growth and early sporulation, We have purified sigma(A) and sigma(H) proteins, and characterized their properties. The genes encoding sigma(A) or sigma(H) were separately cloned into an expression vector under the control of T7 promoter. Both proteins were overproduced in Escherichia coli BL21(DE3) and purified from inclusion bodies after solubilization with guanidine hydrochloride, Antigenicities and N-terminal amino acid sequences of the overproduced proteins were used to identify both proteins. Unlike sigma(A) protein, sigma(H) protein showed a DNA-binding ability. To compare the promoter selectivity of the sigma(A) protein with that of the sigma(H) protein, transcription in vitro of 16 promoters was performed using RNA polymerase holoenzymes reconstituted from a purified core enzyme with either sigma(H) or sigma(A). These holoenzymes correctly recognized each of the cognate promoters; sigma(H)-RNA polymerase recognized sigma(H) promoters but not sigma(A) promoters, and vice versa, A competition experiment for core RNA polymerase using sigma(A) and sigma(H) revealed that sigma(A) had a stronger affinity. We propose that the predicted replacement of a sigma subunit in a holoenzyme from sigma(A) to sigma(H) in vivo at late logarithmic growth phase may require an additional factor, or the modification of a core enzyme or sigma factor.