application): Bacterial DNA-dependent, RNA polymerase (RNAP) is a multiprotein complex consisting of two alpha-subunits, one beta, one beta', and one sigma subunit; the ultimate step of assembly being the alpha2 beta beta' + sigma-alpha2 beta beta' sigma complex holoenzyme. In E. coli, two principal sigma subunits of RNAP, sigma 70 and sigma 38, are responsible for: (1) transcription of genes essential for bacterial growth (sigma 70), and (2) transcriptional regulation of genes essential for survival in the stationary phase of bacterial growth. Thus, it is hypothesized that blocking the ultimate step in RNAP complex assembly could be an efficient method for interfering or arresting the bacterial life cycle. In addition, inhibitors of RNAP complex formation could prove useful in antimicrobial drug resistance. Thus, the Specific Aims of this proposal are to: (1) Perform fine-mapping of structure-function analysis of the sigma binding site within the beta'-sigma assembly region of beta' using a specific ELISA assay utilizing purified sigma-proteins and biotinylated synthetic peptides derived from the sequence of the sigma binding region of beta'. (2) Identify D-peptide high affinity ligands to the beta'-derived sigma binding L-peptides taking advantage of the mirror image phage display technique. (3) Evaluate the ability of selected synthetic sigma-binding L-peptides and beta'-binding D-peptide ligands to inhibit bacterial cell growth in vitro. It is further proposed that Phase II methodology will address the selected analog's ability to treat bacterial infections in vivo.
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