Interaction between RNAP and stringently controlled promoters: 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 response. We found that there is a common functional feature of stringently controlled promoters. The interactions between RNAP and this class of promoters are intrinsically unstable and the stability of the complexes between RNAP and stringently controlled promoters is influenced greatly by the superhelicity of DNA template. In addition, we found that four mutant RNAPs appear specifically to destabilize further the interactions with this class of promoters. The behavior of these RNAP mutants with a supercoiled DNA mimics that of wild-type RNAP with a relaxed DNA for stringently controlled promoters, like """"""""stringent"""""""" RNAPs. From our study, we propose that modulation of the instability of the RNAP-stringent promoter complexes is the key element regulating this class of promoters during the stringent response, and that transcription and the stability of initiation complexes at stringently controlled promoters are coupled through modulation in superhelicity. 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. Starting with the studies on the basis for suppression of a temperature sensitive rpoD800 mutant by several rpoH mutations, a collaboration with Dr. Richard Calendar, Univ. of California, Berkeley, we have developed powerful genetic approaches to studying RNAP and sigma factors interaction. We found that almost all of these rpoH suppressors are defective in binding to core RNAP. These rpoH mutations are located in conserved regions 3, 4.2 and RpoH box of sigma-32. Thus, our study demonstrated that multiple regions on sigma-32 affect core RNAP binding. We also found that some of the rpoH mutations are conditionally lethal in the rpoD+ strain. We took advantage of this phenotype to isolate large numbers of temperature-resistant suppressors of these rpoH mutants. We found that some suppressor mutations are mapped either in the rpoB or rpoC genes, encoding the beta and beta' subunit of RNAP, respectively. Identification of a novel RNA-associated protein, RapA: Using a modified procedure for purification of RNAP, we identified a new RNAP-associated protein named RapA. It is a homolog of the SWI2/SNF2 family of eukaryotic proteins. We found that the RapA protein is an integral component of RNAP. We purified the RapA protein from RNAP to homogeneity and found that RapA forms a stable complex with the RNAP holoenzyme as if it were a subunit of RNAP. In addition, RapA is an ATPase, and the ATPase activity is stimulated when it is bound to RNAP; the latter by itself has little or no ATPase activity in this assay. Thus, RapA interacts with RNAP both physically and functionally.
