We have been studying transcription mechanisms by identifying important domains in RNAP by isolation and characterization of mutant RNAPs that have altered particular functions in transcription, and comparing the biochemical properties of the wild-type and mutant RNAPs at specific steps in the transcription process. In the past year, we have focused on the studies of some defined steps in transcription initiation. 1) Promoter clearance. By analysis of a mutant RNAP that has altered stuttering synthesis at galP2, we have proposed a checkpoint model for promoter clearance. We further demonstrated that the fifth position of the initially transcribed sequence of galP2, which immediately follows a run of Ts, is critical for promoter clearance. The switch between nonproductive and productive synthesis during transcription initiation at galP2 is depending on the rate of incorporation of the nucleotide of RNAP at that critical position. This result indicates that kinetic competition plays an important role in regulating transcription function. 2) The interaction between core RNAP and sigma factor. Initially, we have focused on why some core RNAP mutations including rpoB114 caused hyper-temperature-sensitivity of a major sigma mutant rpoD800. We have found that rpoB114 have reduced the level of the mutant sigma70 peptide in the rpoD800 mutant causing the holoenzyme limiting inside cells. Detailed analysis has found that this core RNAP mutation has reduced rate of synthesis of the sigma70 peptide due to its decreased transcription from the rpoD gene, but it has no effect on the stability of the mutant sigma70 peptide. The mechanism by which these rpoB mutant RNAPs have altered transcription of the rpoD operon will be studied further.
