| Over the past twenty years, RNAs have attracted increasing attention because they are found to have many roles in addition to being transfer RNA, ribosomal RNA, and the mRNA, the transient carrier of genetic information. These include many ribozymes and noncoding regulatory RNAs, such as the famous microRNAs. Group II introns are a class of ribozymes known for their self-splicing ability. This dissertation focuses on the biochemical and structural characterization of two group II introns, B.h.I1 and Ll.LtrB. For B.h.I1, a series of mutant constructs were made to assess the influence of the 5' exon structure on splicing. The results suggested that there is an additional structural element upstream of or encompassing the terminator stem-loop structure that influences the splicing of B.h.I1. At the beginning of this work, group II introns were the only large catalytic RNA without a completely solved three-dimensional structure. To solve the complete tertiary structure of a group II intron, I used Ll.LtrB as my sample molecule and applied a photo crosslinking strategy to obtain distance constraints, which were used for computer-assisted modeling. The resulting structure depicts the conformation of the RNA after intron self-splicing and before exon release. This model lays the foundation for understanding the tertiary structure and inter-molecular interactions of group II introns. The model was published at almost the same time as the first crystal structure of a group II intron, which contains the catalytic site but lacks many periphery helices. The comparison between my model and the crystal structure revealed a discrepancy in the arrangement of domain III. To address this, I carried out a new series of photo crosslinking experiments on the residues in domain III to find more detailed information on its location. The location of domain III was confirmed to be within the scaffold of iv domain I, and a new model was built with the new constraints to provide a more refined representation of the intron helices. This model provides much information on the structure and ribozyme activity of group II introns, and also has implication for the structure of spliceosomal snRNAs because of their structural similarities to group II introns. Overall, this work makes significant contributions to the understanding of ribozyme structures. |
