RNA Catalysis William Scott, Dept. of Chemistry and Biochemistry Ribozmes are RNA-based enzymes whose comparatively recent discovery came as major surprize to the scientific community, as it has always been assumed that only proteins could be enzymes. Researchers in the Scott laboratory are trying to understand how ribozymes work, using X-ray crystallography and other biochemical and biophysical techniques. The potential use of ribozymes as therapeutic agents that target RNA viruses (such as HIV) and pathological mRNAs (such as oncogene transcripts) is well-documented. Although the primary motive for our research 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. (Keywords: RNA molecular biology, ribozyme, structural biology, biochemistry and biophysics, catalysis, the origin of life)

The three-dimensional structures of the hammerhead ribozyme at various points on its reaction pathway.

Currently, we are focusing on four problems:

1. What Drives and What Stabilizes the Conformational Change that Activates Catalysis?

Two significant questions arise from our crystallographically trapped conformational intermediate structure (Murray et al., 1998) that are fundamental to hammerhead ribozyme catalysis:  (a) Does removal of the active site 2’-proton drive this con-formational change, or is the proton abstracted afterwards? and (b) Is there a significant degree of bond formation between the attacking 2’-oxygen nucleophile and the scissile phosphorus?  NMR can augment X-ray crystallography to help answer these two questions. By using 31P and 13C solid-state NMR on appropriately labeled RNA crystals in which the conformational intermediate is again trapped, we plan to assess directly the bonding and protonation states of the attacking nucleophile in the trapped hammerhead ribozyme intermediate.

2.  What is the Transition-State Structure of the Hammerhead Ribozyme?

Although we cannot observe a transition-state directly, we are pursuing three independent approaches to crystallizing a hammerhead ribozyme transition-state or intermediate analogue. The first is to use vanadate to mimic the pentacoordinate transition-state, the second is to incorporate one of several dinucleotide phosphoramidites having pentacoordinated atrane linkages, and the third is to trap an additional intermediate using an enzyme active-site mutation.

3. How Does Hammerhead Ribozyme Catalysis in the Crystal Compare to Catalysis in Solution?

We originally optimized the crystallized hammer-head RNA sequence for X-ray diffraction rather than kinetic prowess.  Comparing ribozyme activity in the crystal to what takes place in solution is therefore not straightforward. Crystallization of a kinetically ideal sequence will help us to clarify these comparisons and will provide an independent ex-perimental test of the mechanistic validity of our intermediate trapping experiments.

4. Can We Design New Catalytic RNAs?