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.

假如。该建议的目的是确定茎环结合蛋白的结构基础（SLBP）调节组蛋白mRNA处理和翻译的功能。以前的生化和遗传研究表明，SLBP是起着必不可少的最重要的跨作用因子通过在3'时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复合物对于招聘和组装多蛋白RNA复合物，调节前MRNA处理，翻译和降解组蛋白mRNA。尽管SLBP存在大量的生化和遗传信息，但分子 SLBP-RNA相互作用的基础尚待阐明。我们将采用多学科的方法在结构上表征了SLBP的RNA结合和处理域（RPD）组蛋白mRNA，使用NMR和质谱法。本提案中描述的实验将补充我的实验室正在进行的功能研究，提供与SLBP有关的机械信息功能和当前提出的模型，以调节体内组蛋白代谢。这该提案的具体目的是1）确定果蝇的结构和动态特性在不使用高分辨率NMR的情况下，SLBP RNA结合和加工域（DSLBP RPD）没有RNA 光谱和FT-ICR H/D交换质谱法，2）确定结构和动态使用高分辨率NMR光谱法DSLBP RPD茎环组蛋白mRNA复合物的特性和FT-ICR H/D交换质谱法，3）表征生物学和生化特性 RNA结合和RNA加工中受损的SSLBP RPD突变体的：4）在结构上表征DSLBP 通过NMR光谱法和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的新信息该生物学重要蛋白的结构/功能关系，也为长期奠定了基础目标是（i）了解组装多蛋白复合物的分子决定因素 3'组蛋白mRNA和（ii）的末端，以开发具有新型特异性的RNA结合结构域作为细胞生物学研究的生物传感器或试剂。