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水平上更好地理解拼接。我们这个项目的目标是研究分支站点以及它如何与第二组内含子的整体架构相互作用和影响。我们的中心假设是，D1-D5结构域的总体结构基本保持一致，随着D6的对接和分支部位与D5活性部位的啮合，有一些局部适应。具体目标目标1：构建伊氏海洋杆菌第二组内含子的D_1~D_5结构域，并对其进行鉴定和结构测定。目的：构建伊氏海洋杆菌第II组内含子的所有结构域(d1-d6)，鉴定其结构，并确定其结构，以分析分支位点与活性位点的其余部分的相互作用以及整个内含子的结构。研究设计这两个目标都将涉及在已建立的、容易结晶的O.ihyensis第二组内含子结构上创造变异，在不变性纯化步骤的情况下使它们结晶，并通过多角度分散实验来确定结构，所有这些都基于已建立的PYLE实验室方案。理查森实验室验证工具将提高晶体结构的准确性，这些工具可通过MolProbity网站获得，适用于RNA。目标1将通过用与从基因组DNA中创建原始构建物相同的PCR编辑方法从构建体中编辑D6来实现，然后人为地切除内含子，因为没有D6它将不能剪接。目标2将需要防止以前移除D6的二级切割反应，并创造条件鼓励它与晶体中其余内含子对接。将尝试较温和的条件进行体外转录和剪接，以减少二次切割。此外，还将提供与外显子结合序列互补的寡核苷酸来填充活性部位，这将限制切割D6片段的机会，并为分支部位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.