Experimental and modelling investigation of the dynamics of an RNA translational operator

This work focuses on the replicase translational operator (TR) stem loop of the RNA MS2 bacteriophage. Single stranded RNA (ss RNA) viruses are important organisms as they represent pathogenic species in all walks of life including a number of clinically important species such as HIV, Marburg Virus and Ebola. MS2 provides a great model system in which to study regulatory pathways affected by RNA folds due to the extensive biochemical and structural data available. In MS2 the TR stem loop acts as an allosteric switch, via a sequence specific recognition event involving the phage coat protein. Binding of the coat protein dimer to TR signals a switch in the life cycle of the phage from replication to assembly and release. Binding of the coat protein dimer leads to translational repression the viral replicase cistron and also leads to an allosteric conformational change in the protein. TR also contains the AUG start codon for replicase gene and thus is involved in the regulatory pathway of the cistron. Thus any insight to how the folds in the RNA effect binding or translational repression are important for understanding the MS2 virus and other ssRNA viruses like it. The folding ensembles present in solution for a series of RNA oligonucleotides that encompass TR have been examined. Single molecule fluorescence assays suggest that these RNAs exist in solution as differentially base paired/stacked states at equilibrium. What emerges from the experimental is data consistent with an unfolding mechanism in which interruptions to the A- form duplex such as the single stranded loop, the free ends and bulged base of TR, act as nucleation points of unfolding. Additionally substitution of a uracil to a cytosine in the loop causes heterogeneity in the folded and unfolded ensembles relative to the wild-type molecule. This data is used to study folding of the RNAs by 'steered' molecular dynamics (MD) simulations. These show remarkable agreement with experimental data and provide atomistic detail on the molecular interactions which take place during the unfolding pathways studied. The adopted conformers for each simulation provide details about hydrogen bonding between the base pairs of the stem and confirm predicted conformers from apparent dye separation in FRET experiments.EThOS - Electronic Theses Online ServiceGBUnited Kingdo