The global objective of the research proposed here is to understand the fundamental question of how a small RNA enzyme, called the hammerhead ribozyme, works. Using static X-ray crystallography, dynamic crystallographic intermediate trapping experiments (a form of time-resolved crystallography), together with a solid-state NMR collaboration and a variety of biochemical approaches, the hypothesis that that the RNA molecule itself, rather than simply acting as a relatively passive scaffold for binding metal ions, actively participates in the chemistry of catalysis, will be tested in a variety of ways. In doing so, an understanding of the relationship between catalytic RNA structure and function (i.e., catalysis) will be obtained.
The specific aims of the program described in this research proposal are: (1) to answer the question of how the hammerhead ribozyme is able to switch its catalytic activity between that of an RNA endonuclease and that of an RNA ligase; (2) to elucidate the forces that drive and stabilize the conformational change known to be required for catalysis in the hammerhead ribozyme structure; (3) to solve the structure of a newly discovered hammerhead ribozyme construct that possesses stabilizing tertiary structural contacts between helical Stems I and II which increase its catalytic rate 1000-fold compared to previously studied hammerhead RNA sequences, and (4) to test our understanding of ribozyme catalysis by designing new catalytic RNAs. Each of these four specific aims is designed to probe, independently, the cleavage mechanism of the hammerhead ribozyme from a variety of viewpoints. The potential use of hammerhead ribozymes as therapeutic agents that target RNA viruses (such as HIV) and pathological mRNAs (such as oncogene transcripts) is well documented. Although our primary motive for the research proposed here is to answer questions of a fundamental scientific nature, it is hoped that the results of these studies will provide practical information to the scientific and medical communities to enable more potent and effective ribozyme-based pharmaceuticals to be developed by others.
| Giambasu, George M; Lee, Tai-Sung; Sosa, Carlos P et al. (2010) Identification of dynamical hinge points of the L1 ligase molecular switch. RNA 16:769-80 |
| Robertson, Michael P; Chi, Young-In; Scott, William G (2010) Solving novel RNA structures using only secondary structural fragments. Methods 52:168-72 |
| Ferré-D'Amaré, Adrian R; Scott, William G (2010) Small self-cleaving ribozymes. Cold Spring Harb Perspect Biol 2:a003574 |
| Lee, Tai-Sung; Giamba?u, George M; Sosa, Carlos P et al. (2009) Threshold occupancy and specific cation binding modes in the hammerhead ribozyme active site are required for active conformation. J Mol Biol 388:195-206 |
| Robertson, Michael P; Scott, William G (2008) A general method for phasing novel complex RNA crystal structures without heavy-atom derivatives. Acta Crystallogr D Biol Crystallogr D64:738-44 |
| Lee, Tai-Sung; Silva Lopez, Carlos; Giambasu, George M et al. (2008) Role of Mg2+ in hammerhead ribozyme catalysis from molecular simulation. J Am Chem Soc 130:3053-64 |
| Martick, Monika; Lee, Tai-Sung; York, Darrin M et al. (2008) Solvent structure and hammerhead ribozyme catalysis. Chem Biol 15:332-42 |
| Martick, Monika; Horan, Lucas H; Noller, Harry F et al. (2008) A discontinuous hammerhead ribozyme embedded in a mammalian messenger RNA. Nature 454:899-902 |
| Martick, Monika; Scott, William G (2006) Tertiary contacts distant from the active site prime a ribozyme for catalysis. Cell 126:309-20 |
| Murray, James B; Dunham, Christine M; Scott, William G (2002) A pH-dependent conformational change, rather than the chemical step, appears to be rate-limiting in the hammerhead ribozyme cleavage reaction. J Mol Biol 315:121-30 |
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