THE HAIRPIN RIBOZYME: STRUCTURE, MUTAGENESIS AND MECHANISM OF ACTION

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The hairpin ribozyme is a catalytic RNA that belongs to the family of small ribozymes. Members of this enzyme class perform a site-specific cleavage reaction that involves nucleophilic attack of a scissile phosphorus that leads to formation of a cyclic 2¿,3¿-phosphodiester and a free 5`-hydroxyl group. Among small ribozymes, the hairpin is unusual in that it does not require divalent metal ions for catalysis. Recent structural and chemical studies have suggested that the reaction proceeds via a general base/acid catalyst mechanism involving conserved bases G8 and A38. Residues contributing to electrostatic stabilization of the oxyphosphorane transition state are also required, but have not been identified fully. The goal of this proposal is to examine the structures of several mutant variants of hairpin ribozyme at the general base G8 position, as well as at the scissile bond (A-1). The applicant¿s lab has crystallized such mutants in the form of a 61-mer synthetic all-RNA construct that diffracted previously to 2.19 Angstroms resolution at beamline A-1 (CHESS). These results resulted in: (i) the highest resolution all-RNA structure of any naturally occurring ribozyme, (ii) identification of the inert 2¿-O-methyl group at the nucleophilic site, and (iii) locating the position of a stabilizing CoHexaamine in the S-turn structural motif (Wedekind et al, Manuscript in Preparation). The advantage of the synthetic hairpin construct is that a variety of non-natural nucleotide bases can be incorporated into the chemically synthesized RNA, which is essentially equivalent to site-directed mutagenesis of a protein. In this study, we have prepared a variety of general base mutants including G8I, G8U, G8(2,6 diaminopurine). In each case, the mutant ribozyme was inactive in solution, but could be rescued by addition of exogenous Mg(II). We believe the Mg(II) fulfills an important electronic stabilization role, and propose the collection of data sets in the absence and presence of (electron dense) Mn(II) in order to assess the conformational changes resulting from mutagenesis and to locate the metal binding site(s). In addition, mutants that effect the ring pucker at the scissile position have been created including: 2`-deoxy(A-1), 2`-amino(A-1), and 2`-hydroxyl(A-1). These mutants represent non-cleavable analogs of the substrate, which in the context of the G8 mutants should provide insight into the `natural` modes of hydrogen bonding and sugar puckering at the 2`-nucleophilic position. This work requires synchrotron radiation because we are incapable of collecting high-resolution data on the home source (beyond 3.0 A in most cases). High resolution data are critical to assign ribose ring puckers, to locate solvent molecules, and to understand how the ribozyme stereochemistry contributes to its enzymatic function.