TUNING THE CONFORMATIONAL DYNAMICS OF DIVERSE RNAS

RNA molecules, including those that fold into complex structures, are known to adopt high energy excited states via conformational dynamics. Initial insights into RNA structural dynamics began over 7 decades ago. Since then, methodologies to better characterize structural dynamics, and recently, structural features of excited states, have emerged. Subsequently, numerous roles for conformational dynamics in regulating RNA functions have been characterized. In my thesis work, I have applied solution-state NMR spectroscopy and biochemical assays to define functionally consequential structural dynamics. Further, I have demonstrated the importance of the kinetics, the rates underlying structural dynamics, in mediating function. I characterized structural dynamics in mutant and homologous RNAs to understand how subtle or not-so-subtle variations in dynamics can alter the biological outcome. First, I have discovered and characterized how a functionally critical base pair in mosquito borne flavivirus Xrn1-resistant RNAs suppresses its opening dynamics by several orders of magnitudes to confront the host exonuclease Xrn1 and promote viral replication. In collaborations with virologists and computational biologists, I have gained insights into the molecular mechanism that underlies the suppressed opening rate as well as the in-cell consequences of enhancing base pair opening. Second, I have characterized how the opening dynamics of a reverse Hoogsteen base pair in the fluoride riboswitch can be rationally ‘tuned’ by either increasing or decreasing these dynamics. Further functional characterization reveals that alterations in linchpin dynamics affects the riboswitch broadly, both by changing the efficiency of the ligand response and altering termination efficiency in the absence of ligand. Finally, I characterized a translation-regulating fluoride riboswitch to gain an understanding into how the conformational dynamics is differentially adapted to regulate at both the transcriptional and translational level. I found that the translational fluoride riboswitch undergoes a remarkably slow transition from the ‘on’ state to the ‘off’ state, shedding light onto how the riboswitch can function in the absence of the tight temporal regulation inherent to its transcriptional counterpart. Taken together, my work has greatly contributed to the current paradigm of RNA structure-dynamics-function relationship by exemplifying the role of conformational kinetics in regulating RNA function.