ABSTRACTMOLECULAR DYNAMICS IN THE HAIRPIN RIBOZYME: CALCULATIONAL AND EXPERIMENTAL ASPECTSByPatrick Omondi OchiengThe increasing role of RNA therapy in targeting diseases has inspired several RNA studies and especially structural RNA. Of interest to many scientists is how such RNA can perform their work with limited functional groups available to RNA. The structural versatility of RNA seems to underscore the importance of dynamics in performing several functions. Ribozymes are a good example of structured RNA involved in RNA backbone cleavage with a range of strategies. Hairpin ribozyme invokes domain-domain docking to activate the cleavage process. The major loop rearrangements observed upon docking, as well as the kinetically unfavorable docking process, both argue for conformational selection by pre-organization of the catalytically-competent active site of the hairpin ribozyme. In this thesis, we sought to study the behavior of loop A in sampling the docked-like conformation as evidence for conformational selection. We addressed three major aims which involved (i) understanding the dynamics in loop A using molecular dynamics simulation as a tool for assessing conformational sampling (ii) determining the right loop A construct for NMR studies and resonance assignments for structure determination and (iii) elucidation of fast and slow dynamics in loop A using NMR relaxation techniques. In aim 1 (Chapter 2), molecular dynamics simulation was used to determine conformational heterogeneity in RNA based on alternate base-pair formation within a subset of residues in the loop region of domain A of the hairpin ribozyme. Three main conformers and several minor conformations were observed in our simulations as analyzed by the Markov State model analysis. RNA base residues and backbone dynamics played a major role in the conformational heterogeneity and ensemble in loop A. Of the conformations that were sampled, the most populated conformer, AA/CA, closely sampled conformational properties similar to the activated (docked) loop A conformation, suggesting the activating role induced by conformational heterogeneity. In aim 2 (Chapter 3), we determined the suitable loop A construct for NMR studies by NMR secondary structure analysis on various constructs of loop A. Using exchangeable and non-exchangeable NMR experiments we assigned certain specific proton, nitrogen and carbon resonances of loop A for structural determination and relaxation measurements.In aim 3 (Chapter 4), we assessed loop A dynamics using the 13C-NMR relaxation measurements. Fast internal motions in the order of ps - ns timescale were analyzed by Model-free approach using data from 13C R1, R1ρ, and heteronuclear NOE of loop A. Loop A was generally found to be a rigid molecule on this timescale with internal generalized order parameters, S2, of at least 0.9 in the helical regions. Several residues reported correlation times indicative of fast motions on the ps to ns timescale, while a few reported slow exchange in the μs-ms timescale.Our data underscores the importance of fast and slow dynamics in the formation of conformational states with structures similar to the activated form. These conformational variability and structural transitions seem to activate RNA thereby facilitating RNA-RNA interaction via the conformational selection mechanism.
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