UC Berkeley

This thesis addresses several questions regarding the structure details of the proteins involved in sigma 54 dependent gene transcription initiation both in E.coli and Aquifex aeolicus. In order to place each topic in its appropriate context, a general introduction is provided in the first chapter, and supplemented with additional, more detailed introductions in each subsequent chapter. The second chapter reflects the central project of this thesis, the determination of the -24 DNA binding domain of sigma 54 from E.coli using Nuclear Magnetic Resonance spectroscopy. The structure, which is a three-helix fold, was compared with the same domain in the sigma 54 from A. aeolicus. DNA binding affinity with the -24 promoter element was also determined by using native gel.

In the third chapter, I describe the previous biochemical studies of histidine kinase sensor protein 4 from Aquifex aeolicus. Then I present my work towards the structural studies of PAS domain of Hksp4 using X-ray crystallography. Diffraction data with 2.8 Angstrom resolution was analyzed, which showed the completeness of the data is below 90%. Molecular replacement was used to build the initial electron density map with no success due to the low sequence identities between the target protein and the models. Experimental phasing was also tried with no success in growing crystals.

In the fourth chapter, I work on the structure determination of NtrC from Salmonella typhimurium, which is among the very first few proteins studied in the sigma 54 dependent gene transcription initiation process. The initial attempt was to use NMR. Beryllofluride was used to activate the RC domains of NtrC. Without the DNA binding domain, RC domains can not be activated by beryllofluride like the full length. Then X-ray crystallography was used for the full-length NtrC to determine the structure of NtrC in the activated state. However, the studies were complicated by low solubility and protein aggregation problems. No crystals were formed in the activated state.