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制水平上更好地理解剪接。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，结构域D1-D5的整体结构基本保持一致，随着D 6停靠和分支位点与D5活性位点接合而具有一些局部调节。具体目标目标1：构建伊海海洋芽孢杆菌II组内含子D1-D5结构域的构建体，对其进行表征，并确定其结构。目标二：建立Ihyensis海洋芽孢杆菌II组内含子的所有结构域D1-D 6的构建体，对其进行表征，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结构。研究设计这两个目标将涉及创造一个既定的，容易结晶O的变化。Ihyensis II组内含子构建体，使它们结晶而不进行变性纯化步骤，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实验室方案。晶体结构的准确性将通过Richardson实验室验证工具得到提高，这些工具可通过MolProbity网站获得，适用于RNA或为RNA开发。目的1将通过用与从基因组DNA产生原始构建体相同的PCR编辑方法从构建体中编辑D 6，然后人工切除内含子来实现，因为没有D 6内含子将不能剪接。目标2将需要防止先前去除D 6的次级切割反应，并创造鼓励其与晶体中其余内含子对接的条件。将尝试较温和的条件用于体外转录和剪接，以减少二次切割。此外，将提供与外显子结合序列互补的寡核苷酸以填充活性位点，这将限制切割D 6片段的机会并产生用于分支位点对接的适当构型。 公共卫生相关性：前mRNA剪接是一个重要的过程，错误与许多人类疾病有关，包括癌症。虽然人类的剪接是由剪接体而不是II组内含子进行的，但II组内含子与剪接体足够相似，其结构可以提供对剪接体的了解，这已被证明在结构上更难研究。此外，II组内含子本身参与基因组的重塑，包括在多重耐药细菌中转移基因，它们存在于细菌中而不是人类中，使它们成为潜在的抗生素靶标。

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.