Interaction between RNAP and the stringently controlled promoters: In a rapidly growing E. coli cell, the vast majority of RNAP molecules engage in transcribing rRNA and a small set of genes. However, upon starvation, transcription of those genes is dramatically reduced in concert with a rapid accumulation of ppGpp, a process termed stringent response. We have found that a complex formed between an RNAP and a stringently controlled promoter is metastable, and is in a rapid equilibrium among different intermediate steps. The stability of the complex is sensitive to ppGpp, salt and the state of supercoiling. We propose that it is this unique feature of the complex that enables RNAP to respond rapidly to environmental changes during growth. In addition, we have found that several mutant RNAPs have decreased transcription initiation specifically at the stringently controlled promoters. The mechanism by which these mutant RNAPs have altered interaction with the stringently controlled promoters will be studied. Interaction between core RNAP and sigma factors: Interaction between core RNAP and different sigma factors forming a holoenzyme is the first step in transcription initiation. We have studied this interaction by mutational analysis. A rpoH mutation that is defective in binding to core RNAP leads to temperature-sensitive for cell growth. We have isolated several second-site temperature-resistant suppressors. Some of these suppressor mutations are mapped in the rpoBC region encoding the two largest subunits of core RNAP. These suppressors mutations will be further analyzed. Identification of a new peptide that is associated with RNAP: Recently, we have found a new peptide (approximately 110 Kd) that is associated with RNAP during a modified purification procedure. The N-terminal of this protein was sequenced and from the data base it is a member of putative helicase with a similarity to the eukaryotic SNF2 and RAD5 helicase family. Currently ,we are trying to purify this new protein and study its function.
