Promoter selectivity of the Bacillus subtilis RNA polymerase sigmaA and sigmaH holoenzymes.
The sigmaH of Bacillus subtilis directs transcription of a large number of early sporulation genes, whereas the principal sigma factor, sigmaA, is essential for the transcription of the genes for vegetative growth and early sporulation. We have purified sigmaA and sigmaH proteins, and characterized their properties. The genes encoding sigmaA or sigmaH 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 sigmaA protein, sigmaH protein showed a DNA-binding ability. To compare the promoter selectivity of the sigmaA protein with that of the sigmaH protein, transcription in vitro of 16 promoters was performed using RNA polymerase holoenzymes reconstituted from a purified core enzyme with either sigmaH or sigmaA. These holoenzymes correctly recognized each of the cognate promoters; sigmaH-RNA polymerase recognized sigmaH promoters but not sigmaA promoters, and vice versa. A competition experiment for core RNA polymerase using sigmaA and sigmaH revealed that sigmaA had a stronger affinity. We propose that the predicted replacement of a sigma subunit in a holoenzyme from sigmaA to sigmaH in vivo at late logarithmic growth phase may require an additional factor, or the modification of a core enzyme or sigma factor.
Fujita
M
M
Radioisotope Center, National Institute of Genetics, Mishima, Shizuoka, 411-8540, Japan. mafujita@lab.nig.ac.jp
Sadaie
Y
Y
D001412:Bacillus subtilis / Q000201:enzymology*; D001483:Base Sequence; D001667:Binding, Competitive; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012689:Sequence Homology, Nucleic Acid; D012808:Sigma Factor / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D012808:Sigma Factor; D006639:Histidine; C063505:RNA polymerase sigma A; D012321:DNA-Directed RNA Polymerases