The mechanisms of the stringent (nutrient starvation) response: During rapid growth in rich media, most RNA polymerase (RNAP) molecules inside the cell are transcribing a small set of genes, most of which are involved in translational machinery. Under nutrient-starvation conditions, a process termed the stringent response, the expression of the above genes (stringent genes), is dramatically reduced. However, the expression of another set of genes, such as amino acid biosynthetic operons, is minimal in rich media and activated during the stringent response. We have been studying the mechanisms by which the dual aspects of the stringent response are coordinately regulated. Previously, we showed that the initiation complexes of stringent promoters are intrinsically unstable and hypothesized that during the stringent response most RNAP molecules will dissociate from this class of promoters, thus increasing the concentration of free RNAP inside the cell. Moreover, we proposed that the rate-limiting step for the promoters that are positively controlled by the stringent response is RNAP binding, thus, the expression of these genes will be activated during the stringent response. We will continue to test the model and to 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. We have continued to study the function of RapA in transcription. In addition, we have analyzed the expression of the rapA gene under different physiological conditions and studied the interaction between RNAP and the rapA promoter in vitro.
