This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Completion of the human genome project revealed a paucity of genes, but an unexpectedly large number of non-protein-coding (nc)RNAs. For example, there are ~4500 ncRNAs in the mouse genome alone and only a handful have an established molecular function. The hairpin ribozyme is an ncRNA derived from the virions of sub-viral plant pathogens. The biological function of the hairpin ribozyme is to cleave concatenated transcripts to unit length by action of an RNA-mediated, site-specific phosphodiester bond cleavage reaction within its cognate substrate. The hairpin ribozyme represents a model system to study ncRNA structure and function due to its small size, readily detectable catalytic activity and its ability to form well-diffracting crystals. The goal of this proposal is to elucidate the functional groups of the hairpin ribozyme that contribute to its million-fold rate acceleration. Recent kinetic analyses of A38 and G8 abasic hairpin ribozyme variants suggested that catalysis of the native RNA does not proceed through a general acid/base mechanism using A38 and G8 (as previously proposed), but instead uses transition-state stabilization involving electrostatic contributions from specific function groups contributed by the exogenously added rescue bases. In addition, the reaction may function in concert with 1-2 waters that serve as specific acid/base catalysts. The applicant's lab previously demonstrated that waters were present in the hairpin ribozyme active site, consistent with the proposed specific base mechanism (Salter et al. &Wedekind, 2006). For the current proposal, we have generated crystals of a minimal all-RNA hairpin ribozyme in complex with a transition-state mimic (vanadate) that should reveal additional, putative specific acid/base catalysts at high resolution. Similarly, we have prepared crystals of a series of G8 or A38 abasic hairpin ribozymes. All crystals have been pre-screened on our home source in Rochester. Crystals exhibited diffraction ranging from 2.8 to 2.3 Angstroms resolution. Previously we demonstrated marked improvement of hairpin ribozyme diffraction at beamline A-1, leading to the identification of conformational heterogeneity at base U37 and a proposal of U39C gain of function (Alam et al. &Wedekind, 2005). We estimate that we will require ~40 hrs to collect diffraction data, which will encompass a variety of abasic samples with different rescue bases. These structures should reveal the mode of rescue base binding in the pre-catalytic state, as well as the presence of putative catalytic waters in the transition state. These observations will be essential for rational kinetic analysis back in our home lab. Ultimately, the result should drive the field forward by proving structural support for the proposed water-mediated mechanism of action. In addition, a greater understanding of the hairpin ribozyme will provide chemical and structure precedents in the analysis of other ncRNAs. For example, water has been observed in the 50S ribosome peptidyl transfer site, but there is no direct evidence that solvent plays an essential role in catalysis. Finally, we propose to collect data on RNA crystals of a 43-mer that represents the Trp/Amber editing site of the hepatitis-delta-virus (MacElrevey &Wedekind, 2005). These crystals are iodinated samples that will be used for multiple isomorphous replacement. The goal of this study is to elucidate the structural features leading to the selection of the RNA editing site by the protein ADAR1, which is essential to complete the viral life cycle. Collection of derivative data sets will require ~10 hrs.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Biotechnology Resource Grants (P41)
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Cornell University
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