| RNA is a class of macromolecules involved in a variety of biological events. RNAs called ribozymes have catalytic functions similar to protein enzymes. Many RNAs are highly structured and are able to recognize other molecules through tertiary interactions. The goal of my research is to investigate the mechanisms of both ribozyme catalysis and RNA-protein interactions, primarily by examining the three-dimensional structures of ribozymes and RNA-protein complexes.; Group II self-splicing introns catalyze auto-excision from precursor RNA transcripts by a mechanism strikingly similar to that of the spliceosome, an RNA-protein assembly responsible for splicing together the protein-coding parts of most eukaryotic pre-mRNAs. Although primary sequence and secondary structural homologies hint at an evolutionary relationship between group II introns and spliceosomal RNAs, the limited three-dimensional structural information for either system has prevented detailed comparison. Here I describe the crystal structure at 3.0 Å resolution of a 70-nucleotide RNA containing the catalytically essential domains 5 and 6 of the yeast ai5γ group II self-splicing intron. The crystal structure reveals that domain 5 adopts an A-form helix conformation with a two-nucleotide bulge and metal ion binding sites analogous to those of the spliceosomal RNAs. Unexpectedly, the branch-point of domain 6 also consists of a two-nucleotide bulged conformation with a bound metal ion, providing a structural explanation for branch-site choice.; Hepatitis delta virus (HDV) is a human pathogen whose genome consists of a single-stranded, circular RNA. The genomic RNA encodes the only known viral protein, hepatitis delta antigen (HDAg), that binds the highly self-complementary genomic RNA. HDAg is required for replication of the RNA genome, a role mediated by the RNA-protein interaction. Both X-ray crystallographic and biochemical approaches are utilized to investigate the nature of this interaction. My results show that purified, recombinant HDAg does not bind specifically to HDV RNA in vitro. The N-terminal helical domain, rather than the proposed RNA-binding domain, is probably responsible for this nonspecific RNA binding. The proposed RNA-binding domain is apparently disordered and its correct folding and specific RNA binding activity might require additional host factors in vivo. |
