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

描述（由申请人提供）：我们的长期目标是在结构和机械层面上更好地理解拼接。我们这个项目的目标是研究分支位点以及它如何与II组内含子的整体结构相互作用和影响。我们的中心假设是D1-D5结构域的整体结构基本保持一致，随着D6的对接和分支位点与D5活性位点的接合，有一些局部的调整。目的1：构建ihyensis (Oceanobacillus ihyensis) II组内含子D1-D5结构域，并对其进行表征，确定其结构。目的2：构建ihyensis group II内含子所有结构域D1-D6的结构，对其进行表征，并确定其结构，以分析分支位点与其他活性位点的相互作用以及内含子的整体结构。研究设计这两个目标都将涉及在已建立的易结晶的O. ihyensis II组内含子结构上创建变体，在不变性纯化步骤的情况下结晶它们，并通过多角度分散实验确定结构，所有这些都基于已建立的Pyle实验室协议。通过MolProbity网站提供的理查德森实验室验证工具，可以提高晶体结构的准确性，这些工具适合或开发用于RNA。目标1将通过使用与从基因组DNA中创建原始结构相同的PCR编辑方法编辑D6来完成，然后人工切除内含子，因为没有D6它将无法拼接。目标2将需要阻止先前除去D6的二次裂解反应，并创造条件鼓励它与晶体中其余的内含子对接。将在较温和的条件下进行体外转录和剪接，以减少二次切割。此外，与外显子结合序列互补的寡核苷酸将被提供来填充活性位点，这将限制切割D6片段的机会，并为分支位点的对接创造适当的配置。

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. PUBLIC HEALTH RELEVANCE: Pre-mRNA splicing is an essential process, and errors are associated with many human diseases, including cancer. While splicing in humans is performed by the spliceosome rather than group II introns, the group II intron is similar enough to the spliceosome that its structure can provide insight into the spliceosome, which has proved much harder to study structurally. Furthermore, group II introns themselves are involved in the remodeling of genomes, including the transfer of genes among multi-drug-resistant bacteria, and their presence in bacteria but not in humans makes them a potential antibiotic target.