Phosphorylation dependent recognition of a histone mRNA hairpin by SLBP

SLBP 对组蛋白 mRNA 发夹的磷酸化依赖性识别

基本信息

批准号：
7029023
负责人：
WILLIAM F. MARZLUFF
金额：
$ 26.36万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-01-01 至 2009-12-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：该提案的目的是确定茎环结合蛋白（SLBP）功能如何调节组蛋白mRNA处理和翻译的结构基础。先前的生化研究和遗传研究表明，SLBP是最重要的跨作用因子，它通过在复制依赖性组蛋白mRNA的3'末端形成高亲和力复合物，在组蛋白mRNA代谢中起着至关重要的作用。 SLBP/RNA复合物对于调节组蛋白mRNA的MRNA前处理，翻译和降解的多蛋白RNA复合物的募集和组装很重要。尽管SLBP存在大量的生化和遗传信息，但SLBP-RNA相互作用的分子基础仍有待阐明。我们将采用一种多学科的方法来使用NMR和质谱法来表征SLBP的RNA结合和加工域（RPD），无论是自由和与组蛋白mRNA的结合。本提案中描述的实验将补充我的实验室中正在进行的功能研究，提供与SLBP功能有关的机械信息，并测试有关SLBP如何调节体内组蛋白代谢的当前建议模型。该提案的具体目的是1）确定果蝇SLBP RNA的结构和动态特性，使用RNA使用高分辨率NMR光谱和ft-icr H/D交换质谱量，使用较高的NMR频谱和动态特性，在RNA缺乏RNA的情况下（DSLBP RPD）确定RNA的结构和动态特性。 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 SLBP的ELF4G。这些研究将提供有关该生物学重要蛋白质的结构/功能关系的新信息，并为长期目标奠定了基础，即（i）了解在组蛋白mRNA的3'末端组装多蛋白络合物的分子决定因素和（ii），以开发具有新型特异性的RNA结合域，这些特异性可用于生物体或经过生物学研究。