Item Details

Print View

Mechanism and Regulation of Transcription Factor Dynamics

Viswanathan, Ramya
Format
Thesis/Dissertation; Online
Author
Viswanathan, Ramya
Advisor
Nakamoto, Robert
Foltz, Daniel
Auble, David Auble
Smith, Jeff
Abstract
Transcription regulation plays a critical role in growth, development and differentiation. The TATA-binding protein (TBP) is a major target for transcriptional regulation. Mot1, a Swi2/Snf2-related ATPase, dissociates TBP from DNA in an ATP dependent process and thereby regulates the binding dynamics of TBP globally. In order to gain a better understanding of the interaction between Mot1 and TBP, we combined X-ray crystallography and electron microscopy structures of Mot1-TBP complexes with biochemical studies. We find that the horseshoe shaped N-terminus of Mot1, consisting of a spring-like array of HEAT repeats, wraps around and grips TBP. We used a FeBABE-mediated hydroxyl radical cleavage assay to map the domains of Mot1 that interact with promoter DNA. Using this assay, we found first that the Mot1 ATPase domain interacts with DNA upstream of the TATA box. Furthermore, using the ATP analog, ADP-AlF 4 , we trapped a putative transition-state complex in which the ATPase domain has undergone a conformational change such that an additional domain is engaged with DNA and the complex is primed for TBP-DNA dissociation. Taken together, this suggests a translocation-based mechanism for TBP-DNA displacement by Mot1. In addition, we modified the widely used chromatin immunoprecipitation (ChIP) assay to study the kinetics of transcription factors binding to chromatin. Using the wellstudied transcription factor Gal4 as an example, we showed that the time dependence of formaldehyde crosslinking can be used to extract in vivo on-rates, offiii rates, occupancies, and residence times of Gal4 at a single locus. Thus, this assay has the potential to provide systematic and quantitative measures of chromatin binding by transcription factors in vivo at unprecedented spatial and temporal resolution. Note: Abstract extracted from PDF text
Language
English
Published
University of Virginia, Department of Biochemistry and Molecular Genetics, PHD, 2012
Published Date
2012-12-01
Degree
PHD
Collection
Libra ETD Repository
In CopyrightIn Copyright
▾See more
▴See less

Availability

Read Online