Insight into ribozyme structure and function using conformationally-restricted RNA: Applications to x -ray crystallography

This thesis describes efforts toward understanding the catalytic mechanism of RNA enzymes (ribozymes) using a multidisciplinary approach. Ribozymes carry out key reactions in biology including phosphoryl transfer and peptide bond formation. In order to carry out their function, ribozymes fold into complex tertiary folds much like protein enzymes. However, in contrast to protein enzymes, RNA is prone to misfolding in solution because of stable base pairing interactions. As a result, one of the major challenges facing RNA biochemists is to solve high-resolution structures of ribozymes that reflect the active, or catalytically-relevant, conformation. In order to reduce the conformational heterogeneity that is inherently associated with RNA, two strategies have been used to facilitate native and homogeneous ribozyme folding. The Hepatitis Delta Virus (HDV) and the lead-dependent ribozyme, which are both implicated in human diseases, have been re-engineered to facilitate mechanistic and structural analyses on the catalytically-relevant conformation.;In the first project, a fast-folding variant of the genomic Hepatitis Delta Virus (HDV) ribozyme has been designed and crystallized for high-resolution structure determination (Chapter 2). By solving the structure of the HDV ribozyme prior to its self-cleavage reaction, the precise role of nucleobase and metal ion participation in the catalytic mechanism may be resolved. In a previous study by another group, an X-ray crystal structure of the pre-cleavage ribozyme was solved; however, several features in this structure are inconsistent with biochemical results. In addition, the structure was solved by replacing a functionally-important cytosine at position 75 (C75) with a non-reactive uridine. Although this modification was chosen to capture the ribozyme in a pre-cleavage conformation, the crystal structure reveals that this mutation alters the architecture of the active site.;The HDV ribozyme variant described in this thesis was specifically designed to address differences between structural and functional data. In addition to incorporating mutations that remove misfolded states, this new construct also includes a native C75. C75 has been shown to function as either a general acid or general base and determining its location in the active site will be an important step toward assigning its exact function. In our study, substitutions were made at the 2'-OH nucleophile in order to prevent the cleavage reaction and maintain the catalytic core. Moreover, by using a ribozyme variant that is less susceptible to misfolding, the use of an RNA-binding protein that was previously used as a crystallization aid could be avoided. Crystals have been obtained for an all-RNA pre-cleavage HDV ribozyme that diffract to 3.0 A resolution. Initial electron density maps show global features that resemble the post-cleavage HDV ribozyme; however, the map does not provide sufficient clarity for unambiguous model building. Additional heavy atom derivative data are needed to overcome the phase ambiguity and to generate a map that is interpretable at the nucleotide level. Efforts toward the structure determination of the pre-cleavage HDV ribozyme are described in Chapter 3.