| The molecular basis for structural cooperativity in large RNAs, the mechanical mechanism by which tertiary interactions work together to influence each other's stability in large RNAs, is difficult to elucidate. The units of a cooperative network within a macromolecule, specific residues, hydrogen bonds, structural units, may be identified in ensemble studies, but the structural effects of these components on global behavior only becomes obvious when one looks at the unaveraged behavior of single molecules.;Here we describe the foundational work for understanding the structural basis for cooperativity in the 260 nt catalytic domain of the RNA component of RNase P from Bacillus stearothermophilus, Cthermo . Single molecule FRET was used to characterize the equilibrium structural fluctuations of Cthermo along its highly cooperative folding path. At low [Mg2+], < 0.1 mM, Cthermo molecules exhibit two-state dynamics described by a double exponential, while at high [Mg 2+], > 0.1 mM, molecules sample multiple states with persistent memory that suggests the presence of stable kinetic traps or alternative folds. The equilibrium behavior of this "wild-type" ribozyme supplies the baseline structural and dynamical framework for mutational and non-equilibrium studies.;Analogous to previous work in which the catalytic domain of RNase P RNA from the mesophilic Bacillus subtilis was converted to thermophilic-like behavior [Fang et al. 2003 J Mol Biol 330:177], we studied three mutants of Cthermo in which one of three key structural motifs, J, P15.1, and P1, a minimal set of "hot spots" for cooperativity that were identified as required for the meso-to-thermo conversion in Fang et al., were swapped for their mesophilic homologs. Preliminary results suggest that in the context of the thermophilic ribozyme, the thermo J motif is essential for cooperative behavior, while the meso and thermo P15.1 and P1 motifs are interchangeable at meso temperatures. Additional tools for single molecule cooperativity studies are also described, including preliminary steps towards relocating the FRET pair in the RNA structure and using periodic Mg 2+ pulses to study the connectivity of states and the structural fluctuations away from the equilibrium basins. Periodic Mg2+ pulse experiments are conducted under conditions in which the RNA structure is not allowed sufficient time to equilibrate during a pulse interval, and therefore the ensemble of structures populated yield EFRET dyamics that are different from equilibrium dynamics. The pulse period length influences the period of subensemble-level oscillations in the RNA response to a Mg 2+ drop, and per-equilibration at constant [Mg2+] indicates the timescale for relaxation towards the non-equilibrium steady-state ensemble that is present under pulsing conditions. |
