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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：
7931205
负责人：
WILLIAM F. MARZLUFF
金额：
$ 16.26万
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：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2010-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7931205
关键词：
Address Affinity Baculoviruses Base Sequence Binding Binding Proteins Biochemical Biochemical Genetics Biological Biology Biophysics Biosensor C-terminal Calorimetry Cells Complement Complex Computational Biology Computational Technique Cyclins Drosophila genus Drosophila melanogaster Drosophila stem-loop binding protein Goals Histones Human Laboratories Mass Spectrum Analysis Messenger RNA Metabolism Modeling Molecular Mutation N-terminal NMR Spectroscopy Nature Nucleotides Peptide Initiation Factors Phase Phosphorylation Play Positioning Attribute Process Property Protein Dynamics Proteins RNA RNA Binding RNA Processing RNA Recognition Motif RNA-Binding Proteins Reagent Recruitment Activity Regulation Regulatory Element Research Design Research Personnel Resolution Role Serine Site Solutions Specificity Structure Structure-Activity Relationship Testing Titrations Trans-Activators Translation Process Translations base in vivo insight mRNA Precursor mutant novel protein complex protein degradation protein function protein protein interaction protein structure research study stem tool

项目摘要

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.

提供了本提案的目的是确定茎环结合蛋白如何（SLBP）的功能是调节组蛋白mRNA的加工和翻译。以前的生化和遗传研究表明，SLBP是一种最重要的反式作用因子，通过在3'端与保守的茎环形成高亲和力复合物在组蛋白mRNA代谢中的作用复制依赖性组蛋白mRNA的末端。SLBP/RNA复合物对于募集和调节前mRNA加工、翻译和降解的多蛋白质-RNA复合物的组装组蛋白mRNA尽管SLBP存在丰富的生化和遗传信息，但其分子生物学特征是， 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，在结构上表征游离和结合SLBP的RNA结合和加工结构域（RPD），组蛋白mRNA，使用NMR和质谱。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，并测试目前提出的模型，SLBP如何调节组蛋白体内代谢。的该提案的具体目标是：1）确定果蝇的结构和动力学特性使用高分辨率NMR在不存在RNA的情况下的SLBP RNA结合和加工结构域（dSLBP RPD）光谱和FT-ICR H/D交换质谱，2）确定结构和动力学使用高分辨率NMR光谱的dSLBP RPD茎环组蛋白mRNA复合物的性质和FT-ICR H/D交换质谱; 3）表征生物学和生化特性的sSLBP RPD突变体在RNA结合和RNA加工中受损，4）结构表征dSLBP 通过核磁共振光谱法测定RPD突变体，以及5）为序列特异性识别提供结构基础翻译起始因子AD 2和eIF 4G的表达。这些研究将提供新的信息，结构/功能的关系，这种生物重要的蛋白质，也奠定了基础，长期目标是：（i）了解多蛋白质复合物组装的分子决定因素组蛋白mRNA的3'末端和（ii）开发具有新特异性的RNA结合结构域，作为细胞生物学研究的生物传感器或试剂。