Phosphorylation dependent recognition of a histone mRNA hairpin by SLBP

但前提是。 这项建议的目标是确定茎-环结合蛋白如何 (SLBP)功能是调节组蛋白mRNA的加工和翻译。以前的生化和遗传 研究表明，SLBP是最重要的反式作用因子，起着至关重要的作用 通过在3‘端与保守的茎环形成高亲和力的复合体在组蛋白mRNA代谢中的作用 依赖复制的组蛋白mRNAs。SLBP/RNA复合体对招募和 调节前mRNA加工、翻译和降解的多蛋白质-RNA复合体的组装 组蛋白的mRNA。尽管SLBP存在丰富的生化和遗传信息，但分子 SLBP-RNA相互作用的基础仍有待阐明。我们将采取多学科的方法来 从结构上表征SLBP的RNA结合和加工结构域(RPD) 组蛋白mRNA，用核磁共振和质谱分析。本提案中描述的实验将 补充我实验室正在进行的功能研究，提供与SLBP相关的机制信息 功能，并测试目前提出的关于SLBP如何在体内调节组蛋白代谢的模型。这个 该提案的具体目的是：1)确定果蝇的结构和动态特性 利用高分辨核磁共振技术研究无RNA条件下SLBP RNA结合和加工结构域(DSLBP RPD) 光谱学和FT-ICR H/D交换质谱学，2)确定结构和动力学 高分辨核磁共振研究dSLBP RPD茎环组蛋白mRNA复合体的性质 和FT-ICR H/D交换质谱仪，3)表征生物和生化性质 在RNA结合和RNA加工中受损的sSLBP RPD突变体，4)dSLBP的结构特征 RPD突变体的核磁共振谱，以及5)为序列特异性识别提供结构基础 SLBP的翻译起始因子AD2和elF4G。这些研究将提供有关 这种生物重要蛋白质的结构/功能关系，也为长期研究奠定了基础 目标是(I)了解多蛋白质复合体组装的分子决定因素 组蛋白mRNAs的3‘端；和(Ii)开发具有新的特异性的RNA结合域 作为细胞生物学研究的生物传感器或试剂。

PROVIDED. The objective of this proposal is to determine the structural basis of how the Stem-Loop Binding Protein (SLBP) functions to regulate histone mRNA processing and translation. Previous biochemical, and genetic studies have demonstrated that SLBP is the single most important trans-acting factor that plays an essential role in histone mRNA metabolism by forming a high affinity complex with a conserved stem-loop at the 3' end of replication-dependent histone mRNAs. The SLBP/RNA complex is important for the recruitment and assembly of multi-protein-RNA complexes that regulate pre-mRNA processing, translation, and degradation of histone mRNAs. Although a wealth of biochemical and genetic information exists for SLBP, the molecular basis for the SLBP-RNA interaction remains to be elucidated. We will take a multi-disciplinary approach to structurally characterize the RNA binding and processing domain (RPD) of SLBP both free and bound to histone mRNA, using NMR and mass spectrometry. The experiments described in this proposal will complement ongoing functional studies in my laboratory, provide mechanistic information relating to SLBP function, and test currently proposed models as to how SLBP regulates histone metabolism in vivo. The specific aims of the proposal are 1) to determine the structural and dynamic properties of the Drosophila SLBP RNA binding and processing domain (dSLBP RPD) in the absence of RNA using high resolution NMR spectroscopy and FT-ICR H/D exchange mass spectrometry, 2) to determine the structural and dynamic properties of the dSLBP RPD stem-loop histone mRNA complex using high resolution NMR spectroscopy and FT-ICR H/D exchange mass spectrometry, 3) to characterize the biological and biochemical properties of sSLBP RPD mutants impaired in RNA binding and RNA processing, 4) to structurally characterize dSLBP RPD mutants by NMR Spectroscopy, and 5) to provide a structural basis for sequence specific recognition of the translation initiation factors AD2 and elF4G by SLBP. These studies will provide new information on structure/function relationships for this biological important protein and also lay the groundwork for long-term goals which are (i) to understand the molecular determinants for assembly of multi-protein complexes at the 3' end of histone mRNAs and (ii)to develop RNA binding domains with novel specificities that can be used as biosensors or reagents for cell biological studies.