Force manipulation and single molecule FRET of transcriptional regulatory factors

transcription;single-molecule;optical tweezers;Förster Resonance Energy Transfer (FRET);biophysics

Brenner, Michael

关键词: transcription;    single-molecule;    optical tweezers;    Förster Resonance Energy Transfer (FRET);    biophysics;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/42145/Michael_Brenner.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Mechanical tension plays a large role in cell development ranging from morphology togene expression. On the molecular level, the effects of tension can be seen in the dynamicarrangement of membrane proteins as well as the recruitment and activation of intracellularproteins leading to downstream signaling cascades regulating transcription. Forces applied tobiopolymers during in vitro force measurements offer greater understanding of the effects oftension on molecules in live cells, and experimental techniques in test tubes and live cells canoften overlap. Indeed, when forces exerted on cellular components can be calibrated ex vivo withforce spectroscopy, a powerful tool is available for researchers in probing cellularmechanotransduction on the molecular scale. Here we report the effect of peptide length on thetension sensing properties of GPGGA peptide repeats using single-molecule fluorescence-forcespectroscopy. Additionally, we report on the mechanical properties of IκBα, a transcriptionalregulator, and the C-terminal domain of RNA polymerase II. Modification of proteins andpeptides for single-molecule studies was extended to incorporation of unnatural amino acids intoa DNA helicase. Chemical modification of RNA was performed to enable total-internalreflection microscopy of single molecules of the guanine riboswitch aptamer domain, which isinvolved in transcription termination. The combined FRET data support a model in which theunfolded state of the aptamer domain has a highly dynamic P2 helix that switches rapidlybetween two orientations relative to nondynamic P1 and P3. At <<1 mM Mg2+ (in the presenceof saturating guanine) or 1 mM Mg2+ (in the absence of guanine), the riboswitch starts to adopta folded conformation in which loop-loop interactions lock P2 and P3 into place. Anothertranscription terminator, Rho helicase, was studied using single molecule techniques. Ourobservations confirm the tethered-tracking model for RNA-directed Rho motion and suggest arepetitive translocation mechanism involving reversible, step-wise threading of RNA through thecentral Rho cavity in discrete steps, leading to loop formation at the exit side of the cavity. Ourdata reveal that secondary structure and lower UC content of RNA impedes processivetranslocation and results in more backwards motion of Rho helicase. We propose a global modelfor Rho dynamics. Furthermore, these results provide general insights into the mechanisms ofRecA-family helicases and ring-shaped ATPases. Preliminary studies with the humanArgonaute2 nuclease will also be presented.