| Many allosterically regulated transcriptional regulators consist of two independent domains, a regulatory domain and a DNA-binding domain connected by an interdomain linker. Interdomain linkers have been found to actively participate in the allosteric regulation of oligomeric DNA-binding proteins. There are two broad classes of interdomain linkers, helical and non-helical. TetR is a homodimeric transcriptional regulator with a helical interdomain linker. Using DNA-binding assays, the work presented in the second chapter demonstrates that TetR uses primarily an extrinsic mechanism to allosterically regulate its DNA binding affinity. AraC is another homodimeric transcriptional regulator that was previously found to use an extrinsic mechanism to allosterically regulate its DNA binding affinity. AraC uses a non-helical interdomain linker to connect its regulatory and DNA-binding domains. The role of the interdomain linker in regulating the in vivo and in vitro behavior of AraC was examined. Amino acid substitution in the interdomain linker have altered the in vivo phenotype of AraC, and the ability of AraC to bind direct repeat half-sites in vitro. The findings presented in the third chapter demonstrated that the interdomain linker of AraC actively participates in the allosteric regulation of AraC, and suggested that amino acid substitutions in the interdomain linker likely altered the behavior of AraC by modulating the strength of the domain-domain interaction present in the repressing conformation of AraC. |
