Promoter Selectivity of the Bacillus subtilis RNA Polymerase A and H Holoenzymes

1 July 1998

journal article
research article
Published by Oxford University Press (OUP) in The Journal of Biochemistry

Vol. 124 (1) , 89-97
https://doi.org/10.1093/oxfordjournals.jbchem.a022102

Abstract

The σ^H of Bacillus subtilis directs transcription of a large number of early sporulation genes, whereas the principal σ factor, σ^A, is essential for the transcription of the genes for vegetative growth and early sporulation. We have purified σ^A and σ^H proteins, and characterized their properties. The genes encoding σ^A or σ^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 σ^A protein, σ^H protein showed a DNA-binding ability. To compare the promoter selectivity of the σ^A protein with that of the σ^H protein, transcription in vitro of 16 promoters was performed using RNA polymerise holoenzymes reconstituted from a purified core enzyme with either σ^H or σ^A. These holoenzymes correctly recognized each of the cognate promoters; σ^H-RNA polymerase recognized σ^H promoters but not σ^A promoters, and vice versa. A competition experiment for core RNA polymerase using σ^A and σ^H revealed that σ^A had a stronger affinity. We propose that the predicted replacement of a subunit in a holoenzyme from σ^A to σ^Hin vivo at late logarithmic growth phase may require an additional factor, or the modification of a core enzyme or σ factor.

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