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

Abstract

Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique that reports structural and dynamic information over a wide range of biologically relevant timescales at atomic resolution. In this dissertation, a combination of NMR techniques, molecular dynamics (MD) simulations, mutagenesis, and biological assays is implemented to characterize the structural and dynamic properties of the 12 nucleotide single stranded RNA (ssRNA) tail located in the Bacillus subtilis prequeuosine riboswitch aptamer. 13C (R1, R2) spin relaxation and residual dipolar couplings (RDCs) are used in combination with MD studies to gain insights into fast (picosecond to nanosecond) 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 was important 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 in proper riboswitch functioning, and finds that the mutation impacts the time-sensitive functioning of the riboswitch. Overall, this dissertation establishes that ssRNA is capable of forming structured and helical regions, and that ssRNA structure plays an important role in the kinetics of riboswitch function. Further, this dissertation lays out a general approach for assessing the structural and dynamic characteristics of other biologically relevant ssRNA systems.