THE GENE AND PROTEIN SEQUENCES OF YEAST HEXOKINASE AND ESCHERICHIA COLI RECA PROTEIN (RECOMBINATION, SOS RESPONSE)

Part I. The nucleotide and amino acid sequences of hexokinase A and hexokinase B from Saccharomyces cerevisiae have been determined. This information now makes possible the construction of atomic resolution models of these proteins, and allows an assimilation of existing chemical, kinetic, and structural data into a model which describes the reaction pathway of this enzyme in detail. In addition to providing a basis for the construction of accurate three dimensional models of these enzymes, the results of this thesis permit comparisons of the primary structures of hexokinase A and hexokinase B to be made, thereby defining areas of dissimilar amino acid sequence which might be responsible for the observed differences in absolute reactivity, substrate specificity, and control by allosteric effectors. The determination of the nucleotide sequences of these genes and their flanking sequences allows analysis of these structures with respect to the DNA sequences of several other genes encoding glycolytic enzymes in yeast. These comparisons reveal similarities in gene structure which have been hypothesized to play a role in the coordinate regulation of the expression of enzymes of the glycolytic pathway.;Part II. recA protein plays a fundamental role in genetic recombination and induction of host SOS functions in E. coli. The results described in this thesis provide a first step towards understanding the molecular details of the recA protein. The amino acid sequence of the wild type protein has been determined (in conjunction with Aziz Sancar of Yale University) to elucidate structural features of the protein which might play a role in the binding of DNA, ATP, and repressors. Several distinct regions have been identified, and are hypothesized to play specific catalytic roles within the enzyme. Additionally, two mutant recA proteins (recA441 and recA430) have been purified and characterized to determine the nature of the amino acid substitutions which cause the characteristic phenotypes of E. coli strains carrying these recA mutations. These substitutions have been analyzed with respect to other studies attemping to define structure-function relationships within the recA protein; in combination, these results allow specific activities of recA to be tentatively assigned to different regions of the enzyme.