Mutational analysis of the Bacillus subtilis RNA polymerase alpha C-terminal domain supports the interference model of Spx-dependent repression.
The Spx protein of Bacillus subtilis exerts both positive and negative transcriptional control in response to oxidative stress by interacting with the C-terminal domain of the RNA polymerase (RNAP) alpha subunit (alphaCTD). Thus, transcription of the srf operon at the onset of competence development, which requires the ComA response regulator of the ComPA signal transduction system, is repressed by Spx-alphaCTD interaction. Previous genetic and structural analyses have determined that an Spx-binding surface resides in and around the alpha1 region of alphaCTD. Alanine-scanning mutagenesis of B. subtilis alphaCTD uncovered residue positions required for Spx function and ComA-dependent srf transcriptional activation. Analysis of srf-lacZ fusion expression, DNase I footprinting, and solid-phase promoter retention experiments indicate that Spx interferes with ComA-alphaCTD interaction and that residues Y263, C265, and K267 of the alpha1 region lie within overlapping ComA- and Spx-binding sites for alphaCTD interaction. Evidence is also presented that oxidized Spx, while enhancing interference of activator-RNAP interaction, is not essential for negative control.
Zhang
Ying
Y
EBS/OGI School of Science & Engineering, OHSU, 20000 NW Walker Rd., Beaverton, OR 97006, USA.
Nakano
Shunji
S
Choi
Soon-Yong
SY
Zuber
Peter
P
D000483:Alleles; D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008958:Models, Molecular; D009876:Operon / Q000235:genetics; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D015398:Signal Transduction / Q000235:genetics; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D012097:Repressor Proteins; D012321:DNA-Directed RNA Polymerases; C113275:RNA polymerase alpha subunit