Investigation of self-splicing Group II intorns (GIIs) from the motochondria of the yeast, Saccharomyces cerevisiae has shown that GII transcripts undergo self-splicing to yield intron lariats and spliced exons. GII and nuclear pre-mRNA introns use similar chemical mechanisms; consequently, analysis of GII reactions will further understanding of nuclear pre-mRNA splicing. GIIs are organized into discrete secondary structure domains; this suggest that each domain has a definite three dimensional shape that forms independenlty and is maintained in the presence of other domains. Assembly of the tertiary structure of the whoe intron depends on specific contacts between domains. Structural study of isolated domains, mapping interdomain contacts, and assessment of the interchangability domains from different GIIs is proposed. This project will determine catalytically critical substrate contacts between Domains 5 (D5) and other domains. D5 is required for both steps of splicing. Mutant derivatives of D5 will be prepared systematically to alter the structure and evaluate the function of D5 quantitatively. These changes will target sites that are highly conserved among D5s. Kinetic parameters and structural requirements for splicing related reactions will be determined. D5 (or derivatives) will be combined with Domain 6 (D6) to analyze their functional cooperation. D6 contains the branch site that attacks the 5' splice site in the first step of splicing. D5 and D6 will be tested with Exon 2 to study the second step of splicing. To identify the site of D5 action, D5 will be prepared with photoactive reagents and crosslinked to the intron. Finally, chemical synthesis will yield enough D5 for direct structural analysis. RNA catalysis is a widespread biological phenomenon; this project offers insight into the chemical mechanism of a unique RNA catalyst. %%% In the cell nucleus of plants and animals, genes are assembled from mosiacs of expressed and intervening sequences by a process called RNA splicing. All cells with nuclei first make long RNA molecules by copying the DNA directly. The cells then cut and join the large RNA molecules to assemble mature messenger RNAs that will be translated in the cytoplasm to express proteins. The process of RNA splicing must be efficient and accurate to allow functional proteins to be produced. The cellular machinery for RNA splicing is extraodinarily complex, to allow for fine control of gene expression in development, brain function, and the immune system. We know that many genetic abnormalities are associated with faulty RNA splicing, so developing rational and effective treatments will require a thorough description of the process of RNA splicing. The RNA splicing machinery is built up with several small RNAs in assemblies with proteins and dozens of additional protein molecules. Because there are so many different parts, it is hard biochemically to analyze RNA splicing from higher cells in detail. This project will analyze a special kind of RNA splicing machinery that operates in the mitochondrion, the energy producing compartment of yeast cells. This special RNA splicing reaction uses the same chemistry as splicing in plant and animal cells, but only RNA molecules are needed in this case. We are studying how two different RNA molecules recognize and bind to each other specifically and then work together to carry out the reaction of splicing. In particular , we will find out what parts of each molecule are needed for splicing and where each molecule touches the other one. We want to produce a very detailed picture of the molecular structure of these two RNA molecules. This work will provide a better understanding of RNA splicing as it occurs in cells and a more precise picture of catalytic strategies that may be used by RNA enzymes. Another benefit of this project will be the possible rational design of antibiotics to prevent growth of yeast and fungus pathogens that afflict humans, animals, and crops plants, since animal cells do not use this special type of RNA splicing that is found in virtually all fungi.
