This project is devoted to defining the ways living cells coordinate metabolic pathways with growth. The system currently studied consists of the pathways to isoleucine and valine in bacteria. These pathways provide good examples of both specific controls (related to isoleucine and valine biosynthesis) and general controls (related to growth rate and state of the cells). It is proposed to examine the significance of the principal form of specific control of the one multicistronic (ilvGMEDA) operon of the ilv gene cluster in Escherichia coli, attenuation of the leader transcript, by making specific changes (site-specific mutagenesis) in the leader region and examining the consequences of these changes. In these experiments, advantage can be taken of the availability of leader regions from five enteric species of which two are sufficiently different from the other three and from each other that there will be more latitude in mutagenic targets than would occur if only the E. coli leader were available. The resulting in vitro-generated mutants will be screened in vitro and with an ilv- lac fusion strain to which an altered leader region can be readily transferred. The altered leaders with their associated promoters will also be inserted into a plasmid in which the cat gene will be under ilv control. Such plasmids will be examined in both multicopy and single-copy states. The mutant leaders will be studied in vitro in a transcription system that allows rigorous comparison with better studied promoters such as rrn and lac. A structure-function analysis of the endproduct-sensitive enzyme, threonine deaminase, will be initiated in which the molecular nature of mutations affecting the regulatory properties of the enzyme will be defined and site-specific mutagenesis will be used to modify the protein.
