Dynamics and allostery in protein-RNA regulation

项目摘要/摘要： 这一建议聚焦于生物学中两个具有广泛重要性的现象： (1)同源寡聚蛋白调节中的变构作用。同源寡聚蛋白广泛存在 在监管系统中比例过高。配体结合部位之间的变构通讯是 对于有效的调节和理解变构偶联的机制至关重要 同源寡聚体的亚基(原单位)对于操纵生物调节至关重要，并且作为一种 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。蛋白质在蛋白质中起着关键作用 结构RNA的适当折叠和组装，使包括核糖体组装在内的功能成为可能， RNA催化和转录调控。RNA折叠问题是重要的，因为局部 单链RNA中碱基配对和堆积相互作用使其能够折叠成许多替代 构象。蛋白质介导的RNA重塑对于理解 调节RNA的功能，但蛋白质可以偏向折叠自由能的机制 风景在很大程度上是一个谜。 我们建议研究同十一聚体(11-mer)形成环的芽孢杆菌Trp RNA结合 衰减蛋白(TRAP)及其与其激活剂配体色氨酸(Trp)的相互作用 靶标、色氨酸前导RNA和抑制蛋白Anti-TRAP。TRAP作为细胞内的一种传感器 色氨酸代谢物(Trp)，可与其11个相同的位点结合，激活它与特定的 Trp操纵子5‘非翻译区的RNA序列。Trp激活的TRAP与RNA结合 导致RNA二级结构重塑，涉及通过流产调节转录 成绩单(终止)。由于其同源-寡聚结构和异构体相互作用，陷阱 是研究各向同性和各向异性机理的一个特殊模型系统。 变构调节。 其目的是(1)确定同源低聚物中的变构通讯机制 配体结合蛋白TRAP；(2)阐明依赖TRAP的RNA折叠在调节中的作用 色氨酸操纵子的转录。这些目标将通过结构性、 热力学、动力学和生化实验，包括核磁共振光谱、量热法、天然 质谱学、共转录化学结构探测、体内和体外生化 化验。

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.