Cellular metabolism includes a multitude of processes essential for the maintenance of life. Studies of metabolism in procaryotes and eucaryotes have clearly established that regulation of enzyme synthesis and the control of activity of preexisting enzymes are two basic mechanisms for adjusting the rates of metabolic reactions in the living cell. The long- term objective of this research is to identify, study and establish novel mechanisms in metabolic regulation at both the protein and the gene level. The inducible biodegradative threonine dehydratase (tdc) operon of Escherichia coli, involved in anaerobic energy metabolism, is an ideal prototype model system to investigate global and operon-specific controls of gene expression and molecular basis of enzyme regulation. A combination of biochemical and molecular genetic techniques will be used to analyze the transcriptional regulation of the tdc genes, characterize their gene products, and examine the molecular architecture of threonine dehydratase and its interactions with allosteric modifiers. Specifically, experiments are proposed to purify the tdcR gene product (an activator protein) and localize its binding site on the tdc promoter for trans-activation of the tdc operon; identify the DNA sequence recognized by the RNA polymerase-sigma54 to deduce its function in mRNA transcription; clone the gene(s) involved in inducer synthesis by in vivo complementation of the inducer-minus phenotype; characterize threonine transport by the tdcA gene product; and isolate mutant forms of threonine dehydratase by site-directed mutagenesis with altered active and regulatory sites to examine the structural requirements for enzyme catalysis and its regulation by cellular metabolites. It is hoped that this integrated knowledge of the genes and proteins in anaerobic threonine degradation will provide new insight into the basic metabolic processes in the living cell and how loss of normal regulatory controls may trigger metabolite imbalance resulting from biochemical and genetic disorders.
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