Establishing the molecular structural basis for branching in pre-MRNA catalysis

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DESCRIPTION (provided by applicant): Our long-term goal is the greater understanding of splicing at the structural and mechanistic levels. Our goal for this project is to examine the branch site and how it interacts with and affects the overall architecture of the group II intron. Our central hypothesis is that the overall structure of domains D1-D5 remains mostly consistent, with some local accommodations as D6 docks and the branch site engages with the D5 active site. Specific Aims Aim 1: To create a construct of domains D1-D5 of the Oceanobacillus ihyensis group II intron, characterize it, and determine its structure. Aim 2: To create a construct of all domains, D1-D6, of the Oceanobacillus ihyensis group II intron, characterize it, and determine its structure to analyze the branch site's interactions with the rest of the active site and the overall intron architecture. Research Design Both aims will involve creating variations on an established, easily crystallizable O. ihyensis group II intron construct, crystallizing them without denaturing purification steps, and determining the structure by multi-angle dispersion experiments, all based on established Pyle laboratory protocols. The accuracy of the crystallographic structure will be enhanced with Richardson laboratory validation tools, available through the MolProbity site, adapted or developed for RNA. Aim 1 will be accomplished by editing D6 out of the construct by the same PCR editing approach used to create the original construct from genomic DNA, then excising the intron artificially since it will not be able to splice without D6. Aim 2 will require preventing the secondary cleavage reactions that have previously removed D6 and creating conditions that encourage it to dock with the rest of the intron in the crystal. Milder conditions will be tried for in vitro transcription and splicing, to reduce secondary cleavage. Also, oligonucleotides complementary to the exon-binding sequences will be supplied to fill the active site, which should limit the opportunity to cleave pieces of D6 and create an appropriate configuration for docking of the branch site.