The long-term objective of this continuing research program is to develop a detailed understanding of the regulation and structure/function relationships of aspartate transcarbamoylase. The cistrons encoding the regulatory and catalytic polypeptides are organized as a bicistronic operon but the mechanism of control of gene expression is uncertain: attentuation, translational coupling to minor charged species of arginyl-tRNA, or trans repressor modulation of transcriptional initiation. These regulatory schemes involve a complex interrelationship between aminoacid biosyntheses and de novo pyrimidine biosynthesis. It is an ideal model system for describing the regulatory logic response to manipulable endogenous pools of related metabolites. The mechanisms of regulatory control will be evaluated by nucleotide sequencing of mutant promoter mutants isolated from promoter/operator lacZ fusions, by comparison with pyrBI promoters from other enteric bacteria, by the creation of specific deletions and site-specific mutations in vitro, by expression in various trans-acting mutations (rpo*, an up-promoter for pyrBI and pyrE, and in specially designed strains that modulate endogenous nucleotide pools), and by expression of various pyrBI operons in heterologous host cells. Furthermore, it is known that pyrBI, pyrE, and pyrF comprise a cooperative regulon which responds to uridylate or uridylate/cytidylate pools and pyrE has a similar attenuator structure. Another objective is to describe the mechanism of this cooperative regulation. In addition, it is clear from preliminary evidence that the details of the regulation of gene expression in other enteric bacteria is varied in subtle ways that may provide additional insight into mechanistic particulars. The second long-term objective of this research is to aid in the description of the structural characteristics involved in defining the catalytic and allosteric control of ATCase. The nucleotide sequence of the regulatory cistron (pyrI) has identified some differences from the published aminoacid sequence and need to be verified. ATCases from other enteric bacteria possess the same architecture, 2(c3):3(r2), yet differ in both catalytic and regulatory characteristics. In addition, the argI cistron encodes a polypeptide for ornithine transcarbamoylase which represents a divergent but evolutionarily related enzyme. It will be possible to probe the functional changes in enzymatic characteristics by comparing deduced aminoacid sequences and predicted secondary structures. Finally, site-specific mutagenesis will allow the manipulation of specific residues.
