AKTA Pure Chromatography System

AKTA 纯色谱系统

• 批准号: 9273230
• 负责人: RUI ZHAO
• 金额: $ 5万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9273230
• 关键词: Active Sites; Biochemical Genetics; Biochemistry; Boxing; Catalytic Domain; Chromatography; Complex; Cryoelectron Microscopy; Crystallization; Crystallography; Data Collection; Disadvantaged; Disease; Electron Microscopy; Electrons; Essential Genes; Eukaryota; Exons; Experimental Designs; Gene Expression; Goals; Guanosine Triphosphate Phosphohydrolases; Health; Heart; Hereditary Disease; Heterogeneity; Introns; Lead; Length; Ligation; Microscopic; Molecular; Nature; Play; Process; Proteins; RNA; RNA Splicing; Reaction; Regulation; Research Personnel; Resolution; Role; Sampling; Site; Small Nuclear Ribonucleoproteins; Sodium Chloride; Spliceosome Assembly Pathway; Spliceosomes; Structure; System; Testing; Time; U5 Small Nuclear Ribonucleoprotein; U5 small nuclear RNA; Virus; detector; experience; functional group; genetic approach; human disease; mRNA Precursor; particle; protein complex; protein structure; reconstitution; research study; symposium; yeast genetics

 DESCRIPTION (provided by applicant): Pre-mRNA splicing is essential for gene expression in all eukaryotes and errors in splicing cause genetic disorders and many other diseases. A thorough understanding of the molecular mechanisms of pre-mRNA splicing has the potential to provide useful approaches for human disease therapy. The splicing of introns is carried out through two transesterification reactions catalyzed by the spliceosome, a large RNA/protein complex composed of five snRNAs and over 100 protein factors. The five snRNAs (U1, U2, U4, U5, U6) and associated proteins form snRNPs, which play important roles in intron recognition, splice site definition, and the splicing reaction. U5 snRNP is a core spliceosomal component that contains U5 snRNA and three protein factors (Prp8, Brr2, and Snu114) that form a stable core even after chaotropic salt treatment. U5 snRNA interacts with both exons and is critical for aligning the exons for ligation in the second catalytic reaction. Prp8 lies in the heart of the spliceosome and is hypothesized to help form/stabilize the active site or even contribute functional groups to the catalytic reaction. Brr2 is a DExD/H-box protein responsible for U4/U6 unwinding, an essential step for spliceosomal activation. Snu114 is the only GTPase in the spliceosome and is thought to regulate the activity of Brr2. Crystal structures of several domains of Prp8 and Brr2 are known, but there is no structure of any full-length protein or U5 snRNA. It is not clear how proteins in U5 snRNP interact with each other, with U5 snRNA, or with the spliceosome catalytic core. The goal of this proposal is to determine the structure and function of components of the U5 snRNP, as well as their complexes with other proteins and RNAs (including those at the spliceosome catalytic core), using a combination of cutting edge cryo-electron microscopy, crystallography, biochemistry, and yeast genetics approaches. A thorough understanding of the structure and function of U5 snRNP will significantly advance our understanding of the molecular mechanisms of pre-mRNA splicing in general.

 描述（由申请人提供）：前体 mRNA 剪接对于所有真核生物中的基因表达至关重要，剪接错误会导致遗传性疾病和许多其他疾病。对前体 mRNA 剪接的分子机制的透彻理解有可能提供有用的方法。用于人类疾病治疗的内含子剪接是通过剪接体催化的两个酯交换反应进行的，剪接体是由五个及以上 snRNA 组成的大型 RNA/蛋白质复合物。 100 个蛋白质因子。五个 snRNA（U1、U2、U4、U5、U6）和相关蛋白质形成 snRNP，在内含子识别、剪接位点定义和剪接反应中发挥重要作用，其中 U5 snRNP 是剪接体的核心成分。即使在离液盐处理后，U5 snRNA 和三个蛋白质因子（Prp8、Brr2 和 Snu114）也能形成稳定的核心。 Prp8 位于剪接体的核心，对于第二次催化反应中的连接外显子的排列至关重要，Brr2 被抢夺以帮助形成/稳定活性位点，甚至为催化反应提供官能团。 /H-box 蛋白负责 U4/U6 解旋，这是剪接体激活的重要步骤，是剪接体中唯一的 GTP 酶。人们认为 Prp8 和 Brr2 的几个结构域的晶体结构是已知的，但没有任何全长蛋白质或 U5 snRNA 的结构。 尚不清楚 U5 snRNP 中的蛋白质如何彼此相互作用、与 U5 snRNA 或与剪接体催化核心相互作用。该提案的目标是确定 U5 snRNP 组件的结构和功能，以及它们与其他蛋白质的复合物。和 RNA（包括剪接体催化核心的 RNA），结合使用尖端冷冻电子显微镜、晶体学、生物化学和酵母遗传学方法。对 U5 snRNP 结构和功能的理解将显着促进我们对前 mRNA 剪接分子机制的理解。