Characterization of TATA Box Binding Protein Interaction with Minicircle DNA

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dc.contributor.advisorKahn, Jason Den_US
dc.contributor.authorByun, Jung Shinen_US
dc.contributor.departmentBiochemistryen_US
dc.contributor.publisherDigital Repository at the University of Marylanden_US
dc.contributor.publisherUniversity of Maryland (College Park, Md.)en_US
dc.date.accessioned2006-02-04T06:37:25Z
dc.date.available2006-02-04T06:37:25Z
dc.date.issued2005-10-05en_US
dc.description.abstractProtein-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.en_US
dc.format.extent31693937 bytes
dc.format.mimetypeapplication/pdf
dc.identifier.urihttp://hdl.handle.net/1903/3060
dc.language.isoen_US
dc.subject.pqcontrolledChemistry, Physicalen_US
dc.subject.pqcontrolledBiology, Molecularen_US
dc.subject.pqcontrolledChemistry, Biochemistryen_US
dc.subject.pquncontrolledTBPen_US
dc.subject.pquncontrolledDNA topologyen_US
dc.subject.pquncontrolledstirrup flatteningen_US
dc.subject.pquncontrolledchromatin remodelingen_US
dc.subject.pquncontrolledprotein-DNA interactionen_US
dc.subject.pquncontrolledminicircleen_US
dc.titleCharacterization of TATA Box Binding Protein Interaction with Minicircle DNAen_US
dc.typeDissertationen_US

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