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Mutational Analysis of the Bacillus subtilis RNA Polymerase C-Terminal Domain Supports the Interference Model of Spx-Dependent Repression

Ying Zhang, Shunji Nakano, Soon-Yong Choi, and Peter Zuber^*

Department of Environmental & Biomolecular Systems, OGI School of Science & Engineering, Oregon Health & Science University, 20000 NW Walker Rd., Beaverton, Oregon 97006

Received 9 February 2006/ Accepted 2 April 2006

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 (CTD). 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-CTD interaction. Previous genetic and structural analyses have determined that an Spx-binding surface resides in and around the 1 region of CTD. Alanine-scanning mutagenesis of B. subtilis CTD 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-CTD interaction and that residues Y263, C265, and K267 of the 1 region lie within overlapping ComA- and Spx-binding sites for CTD interaction. Evidence is also presented that oxidized Spx, while enhancing interference of activator-RNAP interaction, is not essential for negative control.

Supplemental material for this article may be found at http://jb.asm.org/.

Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.

Present address: Department of Microbiology, HanNam University, 133 Ojung-Dong, Taeduk-Ku, Taejon 306-791, South Korea.

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