Chemistry & Biochemistry
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Item Flexibility and Control of Protein-DNA Loops(World Scientific Publishing Company, 2006-10) Kahn, Jason D.; Cheong, Raymond; Edelman, Laurence M.; Mehta, Ruchi A.; Morgan, Michael A.Protein-DNA loops are essential for efficient transcriptional repression and activation. The geometry and stability of the archetypal Lac repressor tetramer (LacI)-DNA loop were investigated using designed hyperstable loops containing lac operators bracketing a sequence-directed bend. Electrophoretic mobility shift assays, DNA cyclization, and bulk and single-molecule fluorescence resonance energy transfer (FRET) demonstrate that the DNA sequence controls whether the LacI-DNA loop forms a compact loop with positive writhe or an open loop with little writhe. Monte Carlo methods for simulation of DNA ring closure were extended to DNA loops, including treatment of variable protein hinge angles. The observed distribution of topoisomer products upon cyclization provides a strong constraint on possible models. The experiments and modeling imply that LacI-DNA can adopt a wide range of geometries but has a strong intrinsic preference for an open form. The flexibility of LacI helps explain in vivo observations that DNA looping is less sensitive to DNA length and shape than would be expected from the physical properties of DNA. While DNA cyclization suggests two pools of precursor loops for the 9C14 construct, single-molecule FRET demonstrates a single population. This discrepancy suggests that the LacI-DNA structure is strongly influenced by flanking DNA.Item Characterization of TATA Box Binding Protein Interaction with Minicircle DNA(2005-10-05) Byun, Jung Shin; Kahn, Jason D; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Protein-induced bending of DNA plays an important role in regulating its transcription, replication, recombination, and packaging into nucleosomes. In particular, many general and gene-specific transcription factors bend DNA. The TATA box-binding protein (TBP) is essential to promoter recognition, and it provides an especially interesting example of dramatic DNA bending. Previous work in our laboratory has shown that TBP bound to strained DNA can induce negative supercoiling, proposed to be the result of untwisting without the compensating writhe provided by the Phe stirrups. The structural proposal makes the clear predictions that TBP lacking the Phe stirrups will induce negative supercoiling under all conditions, and that the mutant may require negative supercoiling in order to bind at all. To test this prediction, we have made the F99A-F116A and F99A-F116A-F190AF207A site-directed TBP mutants that lack the N and C-terminal stirrups. We have characterized the binding of wild type and mutant TBP to linear DNA and to negatively supercoiled minicircles using quantitative hydroxyl radical footprinting, which is the first application for circular DNA, and electrophoretic mobility shift assays (EMSA). The results of the quantitative hydroxyl radical footprinting and EMSA suggest that mutant TBP binds better to the negatively supercoiled minicircle than to the linear DNA. We also observed quite different footprinting patterns for circular versus linear DNA at the TATA box. This indicates that the structure of TBP bound to minicircles may be different than the linear DNA. The equilibrium dissociation constants (Kd) of wild type TBP derived from hydroxyl radical footprinting titrations for the linear DNA and the -1 topoisomers of 203 bp are 11 nM and 3 nM respectively. This suggests that pre-bending of the TATA box enhanced DNA binding. From these observations, we propose that TBP binding to promoters upon gene activation may be enhanced by the topological strain induced in the DNA upon chromatin remodeling.