Determination of the critical protein-protein interactions in transcription systems and in particular between sigma-N and its activator, and the contribution of the sigma-N holoenzyme-activator complex to the DNA strand separation (melting) process are essential for the understanding of the mechanism of transcriptional activation of the RNA polymerase holoenzyme. The sigma-N protein is well suited to analysis as biochemically active domains have been isolated. The overall aims of the proposed work are to: (a) Locate the putative protein-protein interactions between activator and the sigma-N protein, that appear to be critical for activation of RNA polymerase holoenzyme.
(b) Identify promoter DNA-contact regions of the sigma-N
holoenzyme-activator complex during catalysis of open complex formation. (c) Mutate candidate DNA and activator contact regions of sigma-N identified as described above. Screen mutants defective in either a DNA contact during DNA melting and/or activation interaction.
(d) Analyse the DNA-melting process, dependent of the formation of the sigma-N holoenzyme-activator complex.