Single Molecule Flourescence Studies on DExD/H-box Protein:Spliceosome Complexes

项目摘要：多核基因包含内含子，必须通过以下方式从前-mRNA中移除 翻译前的剪接体。剪接体是一台巨型道尔顿大分子机器 由RNA和蛋白质两种成分组成。在此过程中，不利用ATP形成化学键 通过剪接体进行套索合成或外显子-外显子连接。然而，需要三磷酸腺苷的水解。 对于产品形成至关重要的结构重排。这些结构性重组 结果得到明确的、稳定的络合物，这些络合物已被分离并在体外进行研究。目前还不知道是如何做到的 复合体之间会发生转换，但有一种假设是，必需的DExD/H-box蛋白(Prp2，16， 22和43)通过利用ATP水解的能量启动剪接体的结构重组 破坏蛋白质/RNA或RNA/RNA的相互作用。 给定剪接体的~3丙二醛大小，传统的生化分析这些酶是如何 方便拼接是很困难的。在这项研究计划中，单分子荧光(SMF)将被用作 研究DExD/H-box蛋白与剪接体相互作用的新方法剪接体将是 组装在由Prp2缺陷酵母细胞提取物衍生的玻璃表面上。由于没有Prp2， 剪接体将在表面固定的、荧光标记的Pre-mRNA底物上组装，但将 在套索形成之前就停滞了。 PRP酶将使用已公布的程序从大肠杆菌中表达和纯化 使用基因编码的GFP变异体或酮生物素进行荧光标记。之间的相互作用 酶和固定的剪接体将使用多波长SMF显微镜进行可视化。 这些实验将为剪接体催化提供新的见解，而这些剪接体催化是系综方法无法实现的。 通过明确地测试每个DExD/H-box蛋白与 剪接体以及ATP水解区和保守蛋白结构域在剪接体结合中的作用。 相关性：前信使核糖核酸转录本的剪接是基因表达的关键步骤，而前信使核糖核酸转录本的错误 信使核糖核酸剪接与多种癌症有关，包括婴儿脑瘤、乳腺癌、 和视网膜母细胞瘤。理解剪接体如何催化剪接对于理解 在健康和患病的真核细胞中的基因表达。DExD/H-box蛋白的鉴定 利用单分子荧光与剪接体的相互作用将为我们提供新的视角 拼接的化学步骤用整体测量是不可能的。

PROJECT SUMMARY:Mosteukaryotic genes contain introns which must be removed from pre-mRNA by the spliceosome prior to translation. The spliceosome is a mega-Dalton macromolecular machine composed of both RNA and protein components. ATP is not utilized for chemical bond formation during either lariat synthesis or exon-exon ligation by the spliceosome. Nevertheless, ATP hydrolysis is required for structural rearrangements that are essential for product formation. These structural rearrangements result in defined, stable complexes that have been isolated and studied in vitro. It is not known how transitions between complexes occur, but one hypothesis is that essential DExD/H-box proteins (Prp2,16, 22, and 43) initiate structural reorganization of the spliceosome by using the energy of ATP hydrolysis to disrupt protein/RNA or RNA/RNA interactions. Given the ~3 MDa size of the spliceosome, traditional biochemical analysis of how these enzymes facilitate splicing is difficult. In this research proposal, single molecule fluorescence (SMF) will be used as a new method to study interactions between DExD/H-box proteins and the spliceosome. Spliceosomes will be assembled on a derivitized glass surface from Prp2-deficient yeast cell extract. Dueto the absence of Prp2, the spliceosomes will assemble on a surface-immobilized, fluorescently-tagged pre-mRNA substrate but will become stalled prior to lariat formation. The Prp enzymes will be expressed and purified from E. coli using published procedures and fluorescently-labeled using either a genetically-encoded GFPvariant or keto-biotin. Interactions between the enzymes and the immobilized spliceosomes will be visualized using multi-wavelength SMFmicroscopy. These experiments will provide new insight into spliceosome catalysis not attainable with ensemble methods by unambiguously testing the proposed transient associationof each DExD/H-box protein with the spliceosome and the role of ATP hydrolysis and conserved protein domains in spliceosome binding. RELEVANCE:Splicing of pre-mRNA transcripts is an essential step in gene expression, and errors in pre- mRNA splicing have been correlated with a number of cancers including infant brain tumors, breast cancers, and retinoblastomas. Understanding how the spliceosome catalyzes splicing is critical for understanding gene expression in both healthy and diseased eukaryotic cells. Elucidation of DExD/H-boxprotein interactions with the spliceosome using single molecule fluorescence will provide new insight into the chemical steps of splicing not possible with ensemble measurements.