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Doctor of Philosophy (PhD)
Year Degree Awarded
Month Degree Awarded
Lori S Goldner
Craig T Martin
Biological and Chemical Physics
Many RNA interactions in cells occur in the form of loop-loop interactions, also known as a "kissing complex". In the bacterial and viral systems discussed here, there are transiently bound proteins involved that modulate the function of kissing complex. These proteins either stabilize the kissing complex or facilitate its conversion to extended duplex. I studied R1inv-R2inv kissing complex (KC), derivatived from RNAI-RNAII complex of E.Coli. Rop protein is known to stabilize the bent R1inv-R2inv KC against dissociation. The goal was to study structural change of this kissing complex after binding of the stabilizing Rop protein.
In this work for the first time I used the orientation sensitivity of Fluorescence Resonance energy transfer (FRET) to measure an angular change in the structure of R1inv-R2inv kissing complex upon binding of Rop protein. Single-molecular-pair-FRET (spFRET) is often used to study distance fluctuations of single molecules, it is harder to capture angular changes using FRET, because rotational motion of the dyes tends to wash out the angular sensitivity.
The effect of Rop protein on the conformation of the kissing complex is not known. Using fluorescence microscopy techniques we observe a change in twist angle of the KC with protein binding. The eight minimized energy structures reported for R1inv-R2inv KC show a small difference in end-to-end distance and a larger difference in twist and bend angles. From MD simulations I modeled FRET for these eight structures, also for these structures with addition of twist. By comparing the spFRET data with results of this first-principle model, I found the result is consistent with a -25o change in twist angle.
My preliminary work on another kissing complex, Dimerization initiation site (DIS) of HIV-1 retrovirus, is also discussed. Nucleocapsid protein (NCp7) plays an important role in facilitating the kissing complex to extended duplex transition for DIS. My work on DIS kissing complex, was aimed at studying possible intermediates in kissing complex to duplex transition, and investigating the effect of proteins like Rop and NCp7. The construction of the TIRF-FRET instrument, methods for surface passivation, and the RNA sequence design are discussed.
Rahmanseresht, Sheema, "A Quantitative Measurement of Structural changes of RNA kissing complexes using Fluorescence Resonance Energy Transfer" (2016). Doctoral Dissertations. 798.