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JOHNS HOPKINS UNIVERSITY, THE

The long-term objective of this project has been to understand as fully and as deeply as possible, the principles governing the action of the regulatory protein AraC of the L-arabinose operon in the bacterium Escherichia coli and to expand these to elucidate general principles of protein function. The specific aims of the project fell in two general areas, questions related to structure, and questions related to mechanism. We are currently pursuing problems in these areas and we wish to bring them to a conclusion. The scientific aims for the work that will be funded by the supplement are to complete work on the following three questions:1. We can now measure weak protein-protein and domain-domain interactions, and we have measured the interaction between the dimerization and DNA binding domain of AraC. We now want to measure these interactions for two classes of AraC mutants in order to test the predictions of models of AraC action. This will allow us to verify, or extend, or ammend our current model, the light switch mechanism, for AraC action. 2. One of the specific aims of the grant was to understand ion uptake and release that occurs when a protein binds to DNA. While it was known that DNA releases cations when proteins bind, we made the new finding that DNA binding proteins often take up and/or release ions as well in the process of binding to DNA. We have recently observed that the salt concentration dependence of AraC binding to DNA, indicative of ion uptake or release is much different in the presence of arabinose from its dependence in the absence of arabinose. We wish to show that this striking effect is readily explained by or findings and to use this phenomenon to locate a point of residue-residue contact between the two domains of AraC.3. We wish to utilize our newly developed technology for the production of obligate heterodimers to determine whether the binding of one, or of two molecules of arabinose are required to shift AraC from its repressing to its inducing conformation. This determination will indicate whether the induction free energy change of AraC is confined to one subunit, or is distributed between both subunits. Such information is necessary as we test and refine models of AraC mechanism. This project is complete.

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