This research will concentrate on the understanding of the molecular basis of cellular control mechanisms. In particular, to study the actual protein molecules involved in the regulatory process, to examine the molecular basis by which an enzyme can regulate its own activity, and to determine how the cell can regulate the biosynthesis of that particular enzyme. The understanding of cellular regulation will have a great impact on our grasp of cellular differentiation and as a consequence of this, cures for cancer and birth defects may be found. Emphasis will be directed towards three aspects of control of the E. coli pyrimidine biosynthesis pathway, the products of which are necessary for DNA replication. First, the enzyme aspartate transcarbamylase which regulates this pathway by a combination of genetic, metabolic and allosteric control mechanisms; second, the control region of the pyrBI operon; and third, the pyrS gene, a new gene which we recently discovered. The gene-product of pyrS is directly involved in control of this pathway. After the analysis of our current set of mutant versions of aspartate transcarbamylase with single amino acid substitutions is completed, we will use in vitro mutagenesis to generate a group of mutant enzymes with amino acid substitutions at very carefully selected positions. The mutant enzymes will be characterized in order to acquire sufficient information to propose a mechanism for the homotropic and heterotropic interactions of the enzyme as well as the mechanism which governs control of its production. After the genetic characterization of the pyrS gene is completed, we will further characterize the pyrS gene-product. We will also investigate the interaction of the pyrS gene-product with its metabolic effector and its DNA binding site, in order to determine the mechanism by which it exerts control over the pyrimidine pathway.
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