MOLECULAR RECOGNITION DURING PRE-MRNA SPLICING

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 我们建议的总体目标是确定3？剪接位点识别。大多数人类基因转录本是由前-mRNA剪接调控的。剪接体的蛋白质和RNA组分依次识别剪接点，剪接体是一个由100多个蛋白质和5个小核RNA组成的前mRNA剪接机。由必需剪接因子SF1、U2AF65和U2AF35组成的复合体识别前mRNA信号，并将核心剪接机器招募到目标剪接位点。本提案的具体目的是解决以下关于Pre-mRNA剪接关键早期阶段的核心问题：(1)缺乏结构同源的高度保守的SF1结构域的结构是什么？(2)这种SF1结构域的磷酸化通过什么结构手段增强了与U2AF的联系？(3)U2AF通过什么结构手段适应不同的剪接位点？(4)SF1/U2AF复合体与目标剪接位点的三维结构是什么？同步辐射对于实现本提案的目标至关重要，原因包括：(1)波长可调对于多波长异常色散实验是必要的；(2)机器人能力极大地便利了为目标1确定有用晶体所需的广泛筛选；(3)晶体尺寸很小，因此使用常规内部X射线源的衍射非常弱。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The overall goal of our proposed is to determine the structural basis for 3? splice site recognition. The majority of human gene transcripts are regulated by pre-mRNA splicing. The splice sites are sequentially recognized by protein and RNA components of the spliceosome, a pre-mRNA splicing machine composed of more than 100 proteins and 5 small nuclear RNAs. A complex composed of essential splicing factors SF1, U2AF65, and U2AF35 recognizes the pre-mRNA signals and recruits the core splicing machinery to a target splice site. Specific aims of this proposal address the following central questions concerning the critical early stages of pre-mRNA splicing: (1) What is the structure of a highly-conserved SF1 domain that lacks structural homologues? (2) By what structural means does phosphorylation of this SF1 domain enhance association with U2AF? (3) By what structural means does U2AF adapt to diverse splice sites? (4) What is the three-dimensional architecture of the SF1 / U2AF complex with the target splice site? Synchrotron radiation is essential to address the aims of this proposal for reasons including: (i) Tunable wavelengths are necessary for multiwavelength anomalous dispersion experiments; (ii) The robotic capability greatly facilitates the extensive screening required to identify useful crystals for Aim 1; (iii) Crystal size is small and consequently diffraction is prohibitively weak using conventional in-house x-ray sources.