The mechanisms of the stringent (nutrient starvation) response: Under optimal growth conditions, rapidly dividing E. coli cells transcribe a set of genes at a very high rate. These genes engage most of the RNAP molecules in the cell, although they constitute only a small fraction of the genome. In contrast, nutrition-limiting conditions, such as amino acid starvation, cause a rapid accumulation of the guanine derivative, ppGpp, and a dramatic reduction in expression of these genes, a process termed the stringent (nutrient starvation) response. Recently, we have identified a unique feature governing the interaction between RNAP and a class of promoters that are sensitive to the nutrient starvation response. The initiation complexes of these stringent promoters are intrinsically unstable, and can alternate between relatively stable and metastable states depending on the superhelicity of the DNA template. We hypothesized that modulation of the stability of open complexes at these promoters is a regulatory step and proposed a model to link transcription and the stability of the initiation complexes at these promoters. To test this model we have determined roles of the cis and trans elements that regulate the expression of these genes coordinately. In addition, we have continued to isolate and analyze RNAP mutants that altered interaction with stringent promoters to identify the sites in RNAP that are important in the process. Interaction between core RNAP and sigma factors: Since the binding of core RNAP with different sigma factors is, operationally, the first step in transcription initiation, it is a critical step in controlling global gene expression. We have studied this interaction with an emphasis on the role of core RNAP. We have determined the elements that influence the interaction between core RNAP and sigma factors and developed genetic systems to identify the interface between core RNAP and sigma factors. Several sigma mutations that were defective in core RNAP binding conferred temperature sensitive growth phenotype. We have isolated second site mutations in core RNAP that conferred temperature resistant growth phenotype of these sigma mutants. We have continued to characterize these suppressor mutations in core RNAP. Functions of RNAP-associated proteins: Recently, we identified a novel RNAP-associated protein, an ATPase named RapA. The RapA protein is a homolog of the SWI/SNF family of eukaryotic proteins. We showed that RapA forms stable complex with RNAP holoenzyme as if it were a subunit of RNAP, and the ATPase activity of the holoenzyme RNAP-RapA complex is stimulated when compared to the RapA protein alone. We have studied further the interaction between RapA and RNAP (core and holoenzyme) and performed cross-linking experiments to identify the subunit(s) of RNAP that interact with the RapA protein. In addition, we have analyzed the expression of the rapA gene under different physiological conditions aiming at understanding the function of RapA inside the cell, and studied the interaction between RNAP and the rapA promoter in vitro. - Transcription, Transcription Factors, Bacterial genetics, Gene regulation, Biochemistry, prokaryotes, Escherichia coli, protein function, molecular interactions, - Neither Human Subjects nor Human Tissues
