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 have continued to test the model and to determined roles of the cis (sequences in stringent promoters) and trans (Fis, a nucleoid binding protein) elements that regulate the expression of stringent genes. Also, 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. In addition, we have initiated a cellular biology approach to visualize the RNAP distribution in E. coli cells under different physiological environments such as in nutrient rich and poor conditions. 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, and identified several sites in core RNAP that are important for interaction with sigma 70. Functions of RNAP-associated proteins: We found that RapA, identified by us as an RNAP-associated protein and a bacterial homolog of the SWI2/SNF2, activates RNA synthesis by stimulating RNAP recycling. Mutational analyses indicated that the ATPase activity of RapA is essential for its function as a transcriptional activator. Also, we found that the expression of rapA is growth phase dependent, peaking at the early log phase. The growth phase control of rapA is determined at least by one particular feature of the promoter: it uses CTP as the transcription-initiating nucleotide instead of a purine, which is used for most E. coli promoters. SspA, (stringent starvation protein A) is another E. coli RNAP-associated protein, which was described more than two decades ago; its function is still largely unknown. The sequences of SspA are highly conserved and its homologs are implicated in pathogenicity. We found that SspA is required for transcribing bacterial phage P1 late genes both in vivo and in vitro, demonstrating that it is a transcription factor.
