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Made available in DSpace on 2013-02-03T19:17:18Z (GMT). No. of bitstreams: 2 Michael_Brenner.pdf: 48140044 bytes, checksum: 737009cfe6f7c863e8be1a53bd84ce23 (MD5) license.txt: 4065 bytes, checksum: 7b3880bb6f2daecb451ca65aadeab0b2 (MD5) Ha, Taekjip Ha, Taekjip Schulten, Klaus J. Katzenellenbogen, John A. Selvin, Paul R. Brenner, Michael 2013-02-03T19:17:18Z 2013-02-03T19:17:18Z 2017-02-28T10:15:24Z 2018-10-06T09:15:15Z 2012-12 2013-02-03T19:17:18Z 2012-12 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. Item withdrawn by Mark Zulauf (zulauf@illinois.edu) on 2012-12-07T21:39:55Z Item was in collections: University of Illinois Theses & Dissertations (ID: 1) No. of bitstreams: 1 Brenner_Michael.pdf: 47661147 bytes, checksum: 35a44db28ce72dbc5b28b81c6db135c5 (MD5) Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Seth Robbins (srobbins@illinois.edu) on 2013-02-03T19:19:00Z Item is restricted until 2015-02-03T19:18:53Z Restriction data tranferred 2014-07-01T11:33:44-05:00 Original Data Group with Access Administrator Release Date: 2017-02-28 10:16:23 UTC Reason: Author requested U of Illinois access only (OA after 2yrs) in Vireo ETD system; embargo extended another two years to completely closed. Item marked as restricted to the 'Administrator' Group (id=1) by Sarah Shreeves (sshreeve@illinois.edu) on 2014-04-30T15:16:23Z Item is restricted until 2017-02-28T16:16:23Z Limited Restriction Lifted for Item 42092 on 2017-02-28T10:15:24Z. Limited Restriction set for Item 42092 on 2017-10-06T15:15:54Z with date 2018-10-06 by hsherid2@illinois.edu. Limited Restriction set for Item 42092 on 2017-10-06T15:15:58Z with date 2018-10-06 by hsherid2@illinois.edu. Limited Restriction Lifted for Item 42092 on 2018-10-06T09:15:15Z. http://hdl.handle.net/2142/42145 en Copyright 2012 Michael Brenner transcription single-molecule optical tweezers Förster Resonance Energy Transfer (FRET) biophysics Force manipulation and single molecule FRET of transcriptional regulatory factors text Chemistry 1413 Chemistry 0335 University of Illinois at Urbana-Champaign Dissertation Ph.D. PHD:Chemistry -UIUC 10KS0335PHD