Using genetic and biochemical approaches, Dr. Brody proposes to study the role of the T4-coded asiA gene. The 10kDa asiA protein was shown by Audrey Stevens to bind to the sigma-70 subunit of E. coli RNA polymerase, hinder sigma-70 interaction with core enzyme, resulting in inhibition of sigma-70 dependent transcription. Dr. Brody's laboratory has recently identified, cloned, and sequenced the asiA gene, and has overproduced the protein in E. coli. Recent experiments from Brody's lab indicate that the asiA gene is involved in 1- redirecting the E. coli RNA polymerase from T4 early to middle promoters 2- positively activating, in conjunction with the T4 motA protein, E. coli sigma 70 RNA polymerase to form open complexes at middle promoters and 3- possibly aiding T4 sigma 55 in interacting with core RNA polymerase for transcription of the T4 late genes. Recently, Brody's laboratory has been successful in purifying large quantities of active asiA. He now proposes: 1- experiments to investigate the assembly and function of the T4 middle transcription complex. To that end he will use gel filtration and native gel electrophoresis to study the assembly of the complex and reversible protein-protein crosslinking agents to probe for protein contacts in the complex. DNAse I, OH radical and potassium permanganate footprinting of DNA will be used to understand how binding of the various proteins affect each others binding to the promoter. 2- genetic experiments to look at the possible existence of interaction between MotA and asiA and at the interaction of sigma 70 with either T4- coded protein. Sigma 70 deletions and point mutations will be used to map the interaction with asiA. Mutants in sigma-70 resistant to asiA inactivation will be isolated. Physical interactions will be probed by native gel electrophoresis, gel filtration, precipitation with monoclonal antibodies and protease protection experiments. 3- investigate the role of asiA in shutting off early transcription in vivo. 4-investigate the role of asiA in the late transcriptional switch in vitro. 5- determine the effect of gp asiA on sigma factors from many microorganisms to see whether gp asiA could be a useful, specific antibiotic.
