Dynamics and allostery in protein-RNA regulation

蛋白质-RNA 调节的动力学和变构

• 批准号: 9982535
• 负责人: MARK P. FOSTER
• 金额: $ 25万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-01 至 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9982535
• 关键词: 5' Untranslated Regions; Affect; Allosteric Regulation; Bacillus (bacterium); Base Pairing; Binding; Binding Proteins; Binding Sites; Biochemical; Biological; Biological Assay; Biological Models; Biology; Calorimetry; Catalysis; Chemical Structure; Communication; Complement; Coupling; DNA-Directed RNA Polymerase; Data; Drug Design; Drug Targeting; Entropy; Event; Free Energy; Gene Expression Regulation; Genetic; Genetic Transcription; Health; Homo; Human; In Vitro; Investigation; Kinetics; Ligand Binding; Ligands; Mass Spectrum Analysis; Measurement; Mediating; Methods; Modeling; Molecular Conformation; Motion; NMR Spectroscopy; Operon; Pathway interactions; Play; Production; Protein Conformation; Proteins; Protomer; RNA; RNA Binding; RNA Folding; RNA Sequences; Regulation; Regulator Genes; Relaxation; Ribosomes; Role; Site; Structure; System; TYRP1 gene; Technology; Textbooks; Thermodynamics; Titrations; Transcript; Transcriptional Regulation; Tryptophan; Untranslated RNA; Validation; attenuation; base; experimental study; genetic selection; in vivo; inhibitor/antagonist; insight; interest; mutant; protein activation; restraint; role model; sensor; transcription termination

Project Summary/Abstract: This proposal focuses on two phenomena of widespread importance in biology: (1) Allostery in regulation of homo-oligomeric proteins. Homo-oligomeric proteins are widespread and over-represented in regulatory systems. Allosteric communication between ligand binding sites is critical for effective regulation, and understanding mechanisms of allosteric coupling between the subunits (protomers) of homo-oligomers is critical for manipulating biological regulation, and as a means of enabling targeted drug design. However, despite decades of study of allosteric phenomena, the atomic-level linkages between dynamics, thermodynamics and structure remain enigmatic. (2) How proteins remodel noncoding RNA to regulate their function. Proteins play critical roles in proper folding and assembly of structured RNAs, enabling functions that include ribosomal assembly, RNA catalysis and transcriptional regulation. The RNA folding problem is significant because local base pairing and stacking interactions in single stranded RNA enable it to fold into many alternative conformations. Protein-mediated RNA remodeling is particularly important for understanding the function of regulatory RNAs, yet the mechanisms by which proteins can bias the folding free energy landscape are largely a mystery. We propose investigations of the homo-undecameric (11-mer) ring-forming Bacillus trp RNA binding attenuation protein (TRAP), its interactions with its activator ligand, tryptophan (Trp), its regulatory target, the trp leader RNA, and inhibitor protein Anti-TRAP. TRAP serves as a sensor of intracellular tryptophan metabolites (Trp), which can bind its 11 identical sites, activating it for binding to specific RNA sequences in the 5' untranslated region of the trp operon. RNA binding by Trp-activated TRAP results in remodeling of RNA secondary structures implicated in regulating transcription via aborted transcripts (termination). Because of its homo-oligomeric structure and heteromeric interactions, TRAP is an exceptional model system for studying mechanisms of both homotropic and heterotropic allosteric regulation. The aims are to (1) Determine the mechanisms of allosteric communication in the homo-oligomeric ligand binding protein TRAP, and (2) Elucidate the role of TRAP-dependent RNA folding in regulating transcription of the trp operon. The aims will be pursued by a combination of structural, thermodynamic, kinetic and biochemical experiments, including NMR spectroscopy, calorimetry, native mass spectrometry, co-transcriptional chemical structure probing, and in vivo and in vitro biochemical assays.

项目概要/摘要： 这一建议集中在生物学中两个普遍重要的现象： (1)同源寡聚蛋白质调控中的异源性。同源寡聚蛋白广泛存在于 在监管体系中的代表性过高。配体结合位点之间的变构通讯是 关键的有效调控，并了解变构耦合之间的机制， 同源寡聚体的亚基（原聚体）对于操纵生物调节是至关重要的，并且作为 实现靶向药物设计的手段。然而，尽管对变构现象进行了数十年的研究， 动力学、热力学和结构之间的原子级联系仍然是个谜。 (2)蛋白质如何重塑非编码RNA以调节其功能。蛋白质在以下方面起着关键作用： 结构化RNA的正确折叠和组装，实现包括核糖体组装在内的功能， RNA催化和转录调节。RNA折叠问题很重要，因为局部 单链RNA中的碱基配对和堆积相互作用使其能够折叠成许多可选择的结构。 构象蛋白质介导的RNA重塑对于理解蛋白质的功能和作用是特别重要的。 调节RNA的功能，但蛋白质可以偏置折叠自由能的机制 地貌在很大程度上是个谜。 我们提出了研究同源十一聚体（11聚体）环形成芽孢杆菌trp RNA结合 减毒蛋白（TRAP），其与其激活剂配体色氨酸（Trp）的相互作用，其调节 目标，trp前导RNA和抑制蛋白Anti-TRAP。TRAP作为细胞内的传感器， 色氨酸代谢物（Trp），可以结合其11个相同的位点，激活它结合特异性 trp操纵子5'非翻译区的RNA序列。Trp激活TRAP的RNA结合 导致RNA二级结构的重塑，涉及通过中止的转录调节转录， 成绩单（终止）。由于其同源寡聚体结构和异源相互作用，TRAP 是研究同向和异向机理的一个特殊的模式系统 变构调节 目的是（1）确定同源寡聚体中变构通讯的机制 （2）阐明TRAP依赖的RNA折叠在调节细胞凋亡中的作用。 Trp操纵子的转录。这些目标将通过结构、 热力学、动力学和生物化学实验，包括NMR光谱学、量热法、天然 质谱，共转录化学结构探测，以及体内和体外生化 分析。