UC Santa Cruz

As a relatively simple and well-studied molecule, the hammerhead ribozyme is an ideal system to study RNA catalysis and structure. A deeper understanding of the hammerhead catalytic mechanism and the role of divalent ions in catalysis lends support to the exploration of more complex RNA machinery such as the ribosome, and ultimately may assist in the design of new medical therapies. Kinetic study of the hammerhead ribozyme to date has largely derived from bulk assays monitoring millions of molecules. In contrast to such studies, which necessarily report average rates, the magnetic tweezers assay undertaken here reveals the heterogenous characteristics of individual RNA molecules in real time. The crystal structure described in the later portion of this study advances a secondary aim concerning the study of the same full-length Schistosoma Mansoni subject; unmasking the role of physiologically relevant metal ions in full-length hammerhead catalysis.

The single molecule study presented reveals that supercoiled DNA embedded with a nucleolytic ribozyme acts in much the same way as supercoiled DNA. Because the ribozyme is embedded in long DNA handles, the product strands of the ribozyme cannot dissociate, and the ligation rate consequently becomes more significant. This permits us to suggest that in their natural context, hammerheads may well favor the ligated state, and may cleave in response to some form of structural perturbation. This assay also conforms with a growing corpus of evidence suggesting RNA utilizes greater conformational freedom to overcome limited chemical diversity.

The crystal structure described in Chapter 3 represents the highest-resolution structure of any reported hammerhead ribozyme to date (1.55 Å resolution). With this improved resolution, new local rearrangements and hydrogen bonding interactions within the loop/bulge region are clear. In addition, the new structure demonstrates that a divalent metal ion does indeed occupy the A9/G10.1, in addition to two peripheral sites in Stem I and III. Crystal soaking experiments were not able to detect a metal ion at these sites in the 2006 structure. These findings suggest that physiologically relevant divalent ions like Mg2+ may stabilize global folding of the full-length hammerhead and provide charge neutralization during the cleavage reaction.