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Title:Force manipulation and single molecule FRET of transcriptional regulatory factors
Author(s):Brenner, Michael
Director of Research:Ha, Taekjip
Doctoral Committee Chair(s):Ha, Taekjip
Doctoral Committee Member(s):Schulten, Klaus J.; Katzenellenbogen, John A.; Selvin, Paul R.
Department / Program:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
optical tweezers
Förster Resonance Energy Transfer (FRET)
Abstract:Mechanical tension plays a large role in cell development ranging from morphology to gene expression. On the molecular level, the effects of tension can be seen in the dynamic arrangement of membrane proteins as well as the recruitment and activation of intracellular proteins leading to downstream signaling cascades regulating transcription. Forces applied to biopolymers during in vitro force measurements offer greater understanding of the effects of tension on molecules in live cells, and experimental techniques in test tubes and live cells can often overlap. Indeed, when forces exerted on cellular components can be calibrated ex vivo with force spectroscopy, a powerful tool is available for researchers in probing cellular mechanotransduction on the molecular scale. Here we report the effect of peptide length on the tension sensing properties of GPGGA peptide repeats using single-molecule fluorescence-force spectroscopy. Additionally, we report on the mechanical properties of IκBα, a transcriptional regulator, and the C-terminal domain of RNA polymerase II. Modification of proteins and peptides for single-molecule studies was extended to incorporation of unnatural amino acids into a DNA helicase. Chemical modification of RNA was performed to enable total-internal reflection microscopy of single molecules of the guanine riboswitch aptamer domain, which is involved in transcription termination. The combined FRET data support a model in which the unfolded state of the aptamer domain has a highly dynamic P2 helix that switches rapidly between two orientations relative to nondynamic P1 and P3. At <<1 mM Mg2+ (in the presence of saturating guanine) or 1 mM Mg2+ (in the absence of guanine), the riboswitch starts to adopt a folded conformation in which loop-loop interactions lock P2 and P3 into place. Another transcription terminator, Rho helicase, was studied using single molecule techniques. Our observations confirm the tethered-tracking model for RNA-directed Rho motion and suggest a repetitive translocation mechanism involving reversible, step-wise threading of RNA through the central Rho cavity in discrete steps, leading to loop formation at the exit side of the cavity. Our data reveal that secondary structure and lower UC content of RNA impedes processive translocation and results in more backwards motion of Rho helicase. We propose a global model for Rho dynamics. Furthermore, these results provide general insights into the mechanisms of RecA-family helicases and ring-shaped ATPases. Preliminary studies with the human Argonaute2 nuclease will also be presented.
Issue Date:2013-02-03
Rights Information:Copyright 2012 Michael Brenner
Date Available in IDEALS:2013-02-03
Date Deposited:2012-12

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