Investigating the Role of Stingle Stranded RNA Structure on Riboswitch Function and Activity.

Nuclear Magnetic Resonance;RNA;NMR;Residual Dipolar Coupling;Riboswitch;Biological Chemistry;Science (General);Science;Chemical Biology

Eichhorn, Catherine D.Mapp, Anna K. ;

University of Michigan

关键词: Nuclear Magnetic Resonance;    RNA;    NMR;    Residual Dipolar Coupling;    Riboswitch;    Biological Chemistry;    Science (General);    Science;    Chemical Biology;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/94068/ceichhor_1.pdf?sequence=1&isAllowed=y

瑞士|英语

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

【 摘 要 】

Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique thatreports structural and dynamic information over a wide range of biologically relevanttimescales at atomic resolution. In this dissertation, a combination of NMR techniques,molecular dynamics (MD) simulations, mutagenesis, and biological assays isimplemented to characterize the structural and dynamic properties of the 12 nucleotidesingle stranded RNA (ssRNA) tail located in the Bacillus subtilis prequeuosineriboswitch aptamer. 13C (R1, R2) spin relaxation and residual dipolar couplings (RDCs)are used in combination with MD studies to gain insights into fast (picosecond tonanosecond) and slow (up to millisecond) timescale motions. We find that the ssRNA,although highly flexible, adopts a structured, A-form-like conformation within the polyadenine tract. Additionally, the MD simulation shows a similar level of order within the polyadenine tract, with a high level of dynamics at the terminal ends. A domain elongation strategy is applied to decouple internal and overall motions in order to more quantitatively assess dynamics within the ssRNA. We find that the domain elongated ssRNA has similar structural and dynamic properties to the 12 nt ssRNA, and is on average coaxially stacked with respect to the reference helix. We previously identified an A to C mutation in the middle of the polyadenine tract, which was found to destabilize the structural stability of the ssRNA. We hypothesized that ordering of the ssRNA wasimportant for efficient riboswitch function, and that by destabilizing the ssRNA the ability of the prequeuosine riboswitch to terminate transcription efficiently may be reduced. An in vitro transcription assay is developed to address the role of the mutation inproper riboswitch functioning, and finds that the mutation impacts the time-sensitivefunctioning of the riboswitch. Overall, this dissertation establishes that ssRNA is capableof forming structured and helical regions, and that ssRNA structure plays an importantrole in the kinetics of riboswitch function. Further, this dissertation lays out a generalapproach for assessing the structural and dynamic characteristics of other biologicallyrelevant ssRNA systems.