This is a proposal to study the biochemical regulation of enzyme activity during metabolic adaptation and differentiation. Two general questions are posed: (1) what biochemical mechanisms catalyze and regulate the selective inactivation and degradation of enzymes in vivo? and (2) how is the synthesis of enzymes developmentally regulated? These phenomena occur in most cell types and are poorly understood. The experimental system is the differentiating bacterium Bacillus subtilis, in which we have characterized in detail two target enzymes of nucleotide biosynthesis, aspartate transcarbamylase and glutamine PRPP amidotransferase, which are selectively degraded in nutrient-starved (sporulating) cells. The synthesis of these two enzymes is also abruptly shut off prior to their degradation. It is proposed to continue extensive study of the developmental regulation of these enzymes by pursuing the following specific aims: 1. To reconstruct the degradation process in vitro from purified components and to explain the regulation of degradation by the stringent response; 2. To isolate and characterize mutants and clones of genes involved in degradation; 3. To test the hypotheses that degradation of the amidotransferase is preceded by oxidative inactivation of an essential Fe-S cluster; 4. To elucidate the structure and transcription in vitro of the cloned aspartate transcarbamylase (pyrB) gene; 5. To construct mutant forms of the pyrB gene and test their functioning in vivo so as to test hypotheses concerning regulation of pyrB expression; and if resources are adequate 6. To study developmental and nutritional regulation of other genes of the pyr cluster by cloning, sequencing and expression studies with mutant clones.
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