STRUCTURE AND DYNAMICS OF ASIA AND THE ASIA 670 COMPLEX

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The antibiotic consequences of the presence of the AsiA protein product of the asiA gene bacgteriophage T4, are due to its tight interaction with bacteral sigma factors, an example of which is its intaration with sigma 70 of E. Coli. The overall goal of the proposed research is to provide a structural and dynamic platform for understanding, at atamoic resolution, the interation between AsiA and bacterial sigma factors, and as result, to elucidate the mechanism of regulation of bacterial transcription by AsiA. Besides being intrinically relevant to furthering our understanding of the fundamentsls of portein-protein interactions in general, the results are an obligatory prelude to the design of a novel broad spectrumantibioic exploiting AsiA mimicry. The binding of the AsiA protein to the sigma 70 subunit of the E. Coli RNA polymerase is clearly one of the principle factors governing the direction and progression of transcription of the T4 genome. In addition, because of lethal effects on bacteria of overexpression of AsiA, and AsiA-sigma 70 interface could represent the prototype for an exciting novel class of borad spectrum antibiotics and it's target. The research proposed will examine the structure and dynamics of AsiA in solution alone and ocmplexed to the AsiA binding domain of the sigma 70 subunit of the e. Coli RNA polymerase, using primarily nuclear magnetic resonance (NMR) techniques. High resolution structural models fo rthe AsiA protein and its complex with the AsiA binding domain of sigma 70 will be generated using distance geometry/simulated annealing methods and NMR-derieved restraints. Hydrogen exchange and 15N and 13C relatxation techniques will be used to determine the distribution, magnitude, and timescale of motions of the backbone and side chains of AsiA and the AsiA sigma 70 complex. Binding studies and novel high pressure NMR studies wil reveal the site-specific thermodynamics of the AsiA-sigma70 interaction, will provide an intricate account of the static and dynamic events interactions in gerneral, and will provide the critical chemical view necessary for pursuit of structure-based drug design to mimic the antibiotic properties of AsiA.