PHYSICAL AND GENETIC STUDIES OF REGULATORY PROTEINS

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Genetic regulatory processes that modulate transcription initiation rely on specific protein-DNA interactions in both prokaryotic and eukaryotic organisms. Prototypic negative transcriptional regulators in Escherichia coli are lac repressor protein, which prevents initiation of mRNA synthesis for the lactose metabolic enzymes unless lactose is available in the environment, and trp repressor, which represses expression of several different systems in response to intracellular tryptophan levels. The homeotic Ultrabithorax protein serves to specify segmental identity during development in Drosophila by altering transcription of specific genes. These proteins recognize multiple, specific DNA sequences to perform their regulatory function. The lac repressor forms looped DNAs via its two operator sites/tetramer. These loops require tetramer, as dimeric repressor proteins that cannot form loops have been generated. Oligomer formation, subunit assembly and character of the interfaces between lac repressor protomers will be explored using a variety of genetic, chemical and physical methods. The entire repertoire of sequences bound by trp repressor and Ultrabithorax protein will be identified by selection from a random mixture of oligonucleotides, amplification by polymerase chain reaction, and sequencing. To clarify the mode of binding, the stoichiometry of protein:DNA in the complexes formed with oligonucleotides and multi-site DNAs will be evaluated, and the effects of environmental conditions on binding will be measured. Evidence for trp repressor tetramer formation and for Ultrabithorax protein dimer formation suggests the potential for cooperativity in binding tandem sites and/or DNA loop formation via distant sites; these exciting possibilities will be explored using various multi-site DNA constructs with differences in spacing and supercoiling. Clarifying the potential for loop formation in Ultrabithorax action may provide insight into one aspect of the mechanism by which this protein exerts its developmental control, as multiple recognition sites within a regulated transcription unit have been identified. The positions of close interactions between Ultrabithorax protein or its isolated homeodomain and DNA target sites will be explored by DNA modification. Proteolysis and bacterial expression of protein fragments will be employed to explore the domain structure of the Ultrabithorax protein and to identify sites involved in dimer formation as well as to confirm DNA binding sites. The results from these studies will significantly expand our understanding of the structure and function of these essential genetic regulatory proteins.