Date of Award
RNA polymerase, eubacterial sigma, FlgM, sigma factor, anti-sigma
Eubacterial sigma (o) factors are required during the initiation of transcription, where they play key roles in promoter recognition, melting, and transcriptional regulation, sigma is a dissociable subunit of RNA polymerase (RNAP), and sequence-specifically recognizes promoters, but only after associating with RNAP to form the holoenzyme. In the holoenzyme, the major sigma domains (sigma2, sigma3, and sigma4) are spread across the surface, with the promoter binding surfaces solvent exposed and appropriately positioned for simultaneous recognition of the -10 and -35 promoter elements. A -30 residue linker connecting sigma3 and sigma4 is unfolded and threads through the interior of the enzyme. The structure of free sigma is unknown, but biochemical studies suggest that binding to core induces a large conformational change in sigma. The flagellar sigma factor, sigma28 (FliA, sigmaD ), is inhibited by the anti-sigma factor, FlgM, which both prevents and destabilizes its association with core RNAP. N M R analysis of Salmonella typhimurium (St) FlgM suggests that it is unstructured in isolation, but gains structure in the C-terminal half upon binding to sigma28. This work describes the 2.3 A x-ray crystal structure of the sigma^/FlgM complex from Aquifex aeolicus (Aa). Both halves of FlgM are ordered and involved in inhibition of sigma28 by sterically occluding its core binding surfaces, and by stabilizing it in an inactive conformation. Modeling suggests that FlgM can interact with sigma28 in the holoenzyme, explaining its ability to destabilize the sigma28 holoenzyme. The entire sigma28 is ordered, and provides the first structure of an intact sigma factor in the absence of RNAP. There are extensive interdomain contacts, and the promoter binding surfaces of sigma2 and sigma4 are occluded by the sigma-3-sigma4 linker and sigma3, respectively. The sigma3-sigma4 linker forms a bent a-helix, which is incompatible with the holoenzyme conformation, suggesting that a helix-coil transition may accompany holoenzyme formation. Double cysteine mutants of sigma28, predicted to form interdomain disulfides in this conformation, form even in the absence of FlgM, suggesting that free sigma28 adopts this conformation. The disulfide bonded species predominate at equilibrium, indicating that they form in the major solution conformation.
Sörenson, Margareta K., "Structural Analysis of an RNA Polymerase Sigma Factor and Its Anti-sigma: sigma28/FLGM" (2006). Student Theses and Dissertations. 65.