Ribonucleic acid (RNA) is most familiar as the messenger that carries genetic information stored in DNA to the sites where it is translated into the proteins that do the work of the cell. In recent years, however, scientists have realized that RNA molecules that do not encode proteins play a wide variety of roles in biology, and that many of these "noncoding RNAs" take up complex and intricate three-dimensional shapes in order to fill their biological function. In this project, the process by which the well-studied RNA enzyme known as the hairpin ribozyme forms its folded shape will be examined in detail. The results will uncover principles for the processes and driving forces of RNA folding that will be of general applicability to other noncoding RNA molecules and will thus enhance scientific understanding of a variety of fundamental cellular processes. In addition, a standard set of baseline technical parameters (NMR frequencies for atoms in RNA molecules that lack three-dimensional structure) will be generated that will be of use to many workers in the field of RNA biophysics. The lab in which this research will be performed has a strong history in the graduate education of underrepresented groups, and this project will continue and extend these efforts to the undergraduate level via coordination with existing recruitment and retention efforts at Michigan State.
Although the factors underlying secondary structure (helix formation) in DNA and RNA are relatively well understood, the detailed study of the driving forces and mechanisms of the formation of complex tertiary structures in nucleic acids has not kept pace with the great recent advances in RNA structural biology. The active conformation of the hairpin ribozyme is generated by an interaction between two RNA internal loops to generate an intricate docked structure with significant conformational rearrangements. It is hypothesized that this transition is an example of double conformational capture, in which both loops undergo fluctuations to conformation(s) resembling their docked forms and only collisions between loops that are each sampling those forms are productive for recognition. This hypothesis will be tested using NMR spin-relaxation studies of each loop in isolation. Such experiments can identify minor conformers, or "invisible states," sampled by each loop, and the hypothesis predicts that the chemical shifts of these invisible states will resemble those of the docked form. Recently- reported advanced chemical shift calculation methods will be used to estimate the chemical shift changes expected upon transition to a docked-like state. To rule out simple molecular unfolding, a systematic determination of the NMR chemical shifts characteristic of the unfolded state of RNA will be performed, thus producing a reference database likely to be broadly useful to workers in the field. Observed chemical shift changes to the invisible state(s) will be compared to those expected from both fluctuations to the docked-like conformation (as hypothesized) and to simple unfolded states. Finally, advanced molecular mechanics calculations will be used to map out the details of the free-energy landscape implied by the NMR experiments and to verify the existence of energetically accessible trajectories corresponding to the proposed pathways.
