Fret Studies of DNA Binding By Lambda Cro Variants
Date of Award
Ph.D. in Chemistry
Proteins bind to DNA to form switches and circuits that control the flow of genetic information from DNA sequence to the physical components of cells and organisms. The dynamic performance of genetic switches depends on the coupling of the common processes of transcription and translation to protein-specific folding, assembly and binding reactions. For many model circuits protein dimerization and DNA binding are fast relative to the time scale of transcription and translation and thus amenable to modeling by statistical thermodynamics. In these cases, the distribution of proteins between monomers and dimers and between nonspecific and specific DNA complexes can be simply predicted from the measured free energy of each of the states. Lambda Cro is unusual. Dimers of Cro are required to recognize operator DNA and repress transcription, but dimerization is weak compared to DNA binding and slow relative to other processes required for its production and function. Dimerization limits both the equilibrium extent and the kinetic rate of operator binding in vitro. To test the hypothesis that these unusual dimerization characteristics are critical to Cro's regulatory function in living cells, "single-chain dimer" variants of Cro (scCro) that eliminate the slow dimerization step have been characterized. Fluorophore-conjugated, single-cysteine Cro variants have been used to facilitate the investigations of the equilibria and kinetics of DNA binding by fluorescence resonance energy transfer (FRET). Single-chain Cro dimer binds to operator DNA more than 100-fold faster and tighter than wild type Cro. A secondary mutation (scCro(PFPS-AWSG) ) that replaces two proline residues in the context of scCro reduces its equilibrium affinity for operator DNA to a level similar to wild type Cro but binds at a similar rate to scCro. Extrapolation from test-tube to cellular conditions is complicated both by differences in solution conditions and by additional complexity of the intracellular environment. The factor most likely to affect DNA binding in each case is the cellular ionic environment and the presence of a million-fold excess of non-specific DNA binding sites that cumulatively compete with the operator site for Cro binding. To address these differences DNA binding studies as a function of salt and added nonspecific DNA have been undertaken. Kinetic and equilibrium studies of complex formation have been completed by direct FRET measurements and by competition studies with unlabeled proteins and DNA. Specificity ratios obtained for lambda OR3 versus non-specific DNA range from 103 to 106, depending on the Cro variants and the salt concentration. Although the scCro and scCro PFPS-AWSG have similar salt dependences for operator binding, scCro PFPS-AWSG has a significantly higher salt dependence for nonspecific binding. These results will provide inputs for mathematical models of the dynamics and equilibrium extent of repression by Cro variants in living cells.
Wang, Lei, "Fret Studies of DNA Binding By Lambda Cro Variants" (2012). Electronic Theses and Dissertations. 302.