Eukaryotic gene expression is an essential and variegated process that is regulated at multiple levels. The regulation of transcription by DNA binding transcription factors (TFs) is a key level of this process. Our understanding of this regulation has been enabled in large part by the available investigative technologies and techniques in the biochemistry and molecular biology community, including fundamental techniques spanning from in vitro protein:DNA interaction studies to genome sequencing. In efforts to better understand transcriptional regulation, modern extensions and innovations in metholody have been created, such as surface plasmon resonance (SPR), chromatin immunoprecipitation on microarray chip (ChIP-chip) or ChIP coupled to rapid sequencing (ChIP-seq), and protein binding microarrays (PBM). The central importance of multi-protein complexes of TFs in the cell, coupled with the widespread nature of cis-regulatory sequences discovered in the human genome, highlight a great need for technologies and methods able to investigate regulatory DNA-binding protein complexes. This dissertation presents novel optical techniques that are capable of sensitive, high-throughput kinetic and thermodynamic measurements of DNA binding by transcription factor complexes. Total internal reflectance fluorescence protein binding microarrays (TIRFPBM) are a validated and extensible system for multi-protein transcription factor investigation, and rely on the use of evanescent fields in a waveguide-microarray to fluorescently excite transcription factors when bound to DNA targets. Surface-enhanced Raman scattering (SERS) DNA assemblies present a complimentary technique that uses self-assembled, optically active nanoparticle clusters to monitor TF binding in a robust and highly sensitive fashion. Together, these techniques provide a novel and extendible platform for observing and understanding transcription factor complexes during transcriptional regulation, enabling previously difficult-to-approach biochemical investigations.
|Advisor:||Reich, Norbert O.|
|Commitee:||Daugherty, Patrick S., Lew, John, Plaxco, Kevin W.|
|School:||University of California, Santa Barbara|
|Department:||Biomolecular Science and Engineering|
|School Location:||United States -- California|
|Source:||DAI-B 71/05, Dissertation Abstracts International|
|Subjects:||Molecular biology, Biochemistry|
|Keywords:||DNA binding affinity, Protein binding microarrays, Total internal reflectance, Transcription factors|
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