| The work contained in this thesis focused on deciphering how base functional groups and solution components contribute to indirect readout of DNA binding sites by 434 repressor protein. In Chapter 1, we introduce the bacteriophage 434 system. This includes an outline of how 434 repressor recognizes potential DNA binding sites. Additionally, we report what is currently known concerning how solution components mediate DNA/protein interactions.; Chapter 2 tests whether the number of hydrogen bonds, or the presence of an N-2 amine in the center of a DNA binding site, reduces the affinity of repressor for that target DNA. We find that the presence of a central N-2 amine on synthetic DNA binding sites reduced the affinity of repressor for that DNA, compared to its affinity to sites that lack this base functional group. In a series of biochemical assays, we characterize how this central amine reduces the affinity of repressor for a DNA site.; In Chapter 3 we test how cations to mediate the sequence specific recognition of repressor for two DNA binding sites, OR1 and OR3. While changing divalent cations in solution had little effect on the affinity of repressor for either binding site, changing monovalent cations in solution dramatically alters the affinity of repressor for OR1, but not OR3. In a series of tests, we more precisely define how monovalent cations mediate the sequence specific recognition of DNA binding sites by 434 repressor.; In Chapter 4 we describe the association kinetics of repressor for two binding sites, OR1 and OR2. Our data indicate that the rate of association of repressor to OR1 is limited by the time it takes to migrate to this DNA sequence. In contrast, the association rate of repressor to OR2 is limited by an isomerization event at the OR2 site. These results suggest that the differences in base sequence between OR1 and OR2 switch the rate-limiting step in the association of repressor for these DNA's.; Chapter 5 summarizes the results obtained in the previous three chapters, and outlines the significance of the work in this thesis. |
