Group I introns function both as intervening sequences and as transposable elements in fungal mitochondria, in chloroplasts, in bacteria and in the nuclei of some simple eukaryotes. In many instances, the removal of group I introns from precursor RNA (splicing) is self-catalyzed in vitro. This project concentrates on the splicing of a group I intron found in the gene for apocytochrome b (COB) in the mitochondria of bakers' yeast (Saccharomyces cerevisiae). The structure of the fifth intron of the COB gene (bI5) is significantly different from that of the best characterized group I intron. In the test tube, splicing of this intron can occur without proteins. In the cell, however, excision of this intron requires a protein encoded by a nuclear gene called CBP2. This is the only intron in which both autocatalytic and protein-enhanced splicing can be studied in vitro. We will exploit this feature of the system to determine the role of the cbp2 protein in splicing. We will also employ a powerful genetic screening system to study protein-dependent splicing in E. coli. Specific experiments include: modifying the intron to """"""""trim off"""""""" sequences that are not essential for autocatalytic processing; using site-directed mutagenesis to characterize novel features of the RNA structure; studying the binding of the cbp2 protein with the intron RNA; determining how this binding alters the conformation of the RNA; generating mutations which affect the splicing of bI5 in E. coli cells. The objective of this research is to understand the physiologic role of a protein in an RNA-specified reaction. Similar protein/RNA interactions are characteristic of a variety of cellular activities including RNA processing, translation and the transcriptional activation of retroviruses. Because the chemistry of group I splicing is identical to the phosphoryl exchange responsible for the splicing of nuclear introns, the system we are studying may serve as a model for that more complex process. Applications of this research include design of RNA catalysts, development of drugs for control of viral and mycotic infections and appreciation of the genetic mechanisms responsible for health and disease.
| Shaw, L C; Lewin, A S (1997) The Cbp2 protein stimulates the splicing of the omega intron of yeast mitochondria. Nucleic Acids Res 25:1597-604 |
| Shaw, L C; Thomas Jr, J; Lewin, A S (1996) The Cbp2 protein suppresses splice site mutations in a group I intron. Nucleic Acids Res 24:3415-23 |
| Shaw, L C; Lewin, A S (1995) Protein-induced folding of a group I intron in cytochrome b pre-mRNA. J Biol Chem 270:21552-62 |
| Ritchings, B W; Lewin, A S (1992) Mutational evidence for competition between the P1 and the P10 helices of a mitochondrial group I intron. Nucleic Acids Res 20:2349-53 |
| Partono, S; Lewin, A S (1988) Autocatalytic activities of intron 5 of the cob gene of yeast mitochondria. Mol Cell Biol 8:2562-71 |
