Initiation of gene expression in prokaryotes and eukaryotes requires specific factors which interact with RNA polymerase to recruit polymerase to the promoter. Recruitment in prokaryotes is accomplished by sigma factors which recognize specific DNA sequences and deliver polymerase proximal to the start site of transcription. Sigma factors have resisted detailed structure/function analysis for more than 25 years and little definitive information has been elucidated on the precise nature of the sigma factor/DNA recognition event. Preliminary data in this proposal demonstrates that the solution of the three-dimensional structure of the primary sigma factor in E. coli, sigma70, is now at hand. Through the cloning of the closely related factors from two thermophilic organisms, the protein/DNA interaction of sigma70 will be defined in molecular detail. Mutagenic and footprinting analysis will complement structure elucidation to provide a comprehensive view into the sigma70/DNA interaction. In addition, the interaction of the C-terminal region 4 of sigma70 will be further characterized for the transcriptional repressor/anti-sigma factor AsiA of bacteriophage T4 as an example of how region 4 acts as a communication locus for activators and suppressors of prokaryotic genes. In this regard, an unexpected stoichiometry of the region 4/AsiA complex has been identified, leading to the intriguing hypothesis that AsiA acts as a DNA mimic to displace sigma70 from the DNA. Novel approaches to the solution of the protein, protein/DNA and protein/protein complexes are described. The 'analog' of a polymerase recruitment factor from eukaryotes is also to be studied in this proposal. The eukaryotic protein is Rap30, a subunit of TFIIF, which shares sequence and functional homology with sigma70. The Rap30 DNA-binding domain has been identified, its three-dimensional structure and the identification of the protein residues in contact with DNA have all be determined as a part of preliminary data. Unlike sigma70, Rap30 does not possess sequence preferences and does not bind DNA with high affinity. To approach the solution of its three-dimensional structure bound to DNA, novel labeling schemes for multinuclear NMR spectroscopy have been developed, permitting a general solution to the non- specific protein/DNA interaction complex problem. Of greatest interest in this regard is the development of a robust DNA labeling scheme which permits analysis of DNA structure in new ways and is essential to the solution of the complex structure. The resultant structure will provide new insight into the role of Rap30 in DNA wrapping for a eukaryotic pre-initiation complex. A preliminary model of a eukaryotic preinitiation complex is presented.
| Bonocora, Richard P; Caignan, Gregori; Woodrell, Christopher et al. (2008) A basic/hydrophobic cleft of the T4 activator MotA interacts with the C-terminus of E.coli sigma70 to activate middle gene transcription. Mol Microbiol 69:331-43 |
| Lambert, Lester J; Wei, Yufeng; Schirf, Virgil et al. (2004) T4 AsiA blocks DNA recognition by remodeling sigma70 region 4. EMBO J 23:2952-62 |
| Lambert, L J; Schirf, V; Demeler, B et al. (2001) Flipping a genetic switch by subunit exchange. EMBO J 20:7149-59 |
