Factors Stabilizing RNA Structures

描述(申请人提供)：RNA分子折叠成特定的三级结构对许多与基因表达有关的细胞过程非常重要：蛋白质合成机制依赖于复杂的转移和核糖体RNA结构，信使和其他RNA的处理需要特定的结构，转录和翻译的调节取决于RNA序列采用特定结构响应蛋白质或代谢物信号的能力。RNA的化学性质限制了它的结构可能性；负电荷导致对存在的离子类型和浓度的敏感性，以及高度水化导致对中性溶液成分(渗透压)的敏感性。这项拟议研究的长期目标是调查所有可能影响RNA稳定性的自然产生的溶液因素，并确定RNA在体内完成特定功能可能使用的不同策略。具体问题将在三个一般领域中的每个领域进行研究：在基础水平上，我们将继续我们的发现，即一些渗透分子通过影响RNA表面的水化来稳定RNA结构，以获得伴随三级结构形成的RNA水化变化的更详细的图像，并扩大我们对离子的研究，包括与体内相关的有机离子。将详细研究两个不同RNA中不同结构之间的构象开关的动力学和能量学，其中开关控制基因表达，以响应细胞内游离镁离子或小代谢物(所谓的“核糖开关”RNA)水平的变化。这两个RNA使用非常不同的策略来实现稳定的三级结构，并且可能也通过不同的机制发挥作用；这些研究旨在探索功能RNA的这些相反的可能性。核糖开关RNA的物理研究将成为体内研究的跳板，旨在阐明细胞环境中的作用机制，例如，在决定RNA对细胞信号的功能反应时，热力学稳定性如何与折叠的动力学速度平衡，RNA对体内发现的各种离子的依赖，以及细菌细胞因生长条件的变化而引起的离子和渗透压的变化如何影响RNA功能。这些研究将有助于阐明RNA如何在细胞环境中发挥作用，并可能应用于设计阻断关键RNA功能或设计具有特定调节功能的RNA。细菌病原体特别依赖核糖开关RNA在人体内外不同的环境中生存，因此是RNA靶向治疗的候选药物。

DESCRIPTION (provided by applicant): The folding of RNA molecules into specific tertiary structures is important for many cellular processes relating to gene expression: the protein synthesis machinery depends on intricate transfer and ribosomal RNA structures, processing of messenger and other RNAs requires specific structures, and the regulation of transcription and translation depend on the ability of RNA sequences to adopt specific structures in response to protein or metabolite signals. The chemical nature of RNA limits its structural possibilities; the negative charge causes sensitivity to the types and concentrations of ions present, and the high degree of hydration induces sensitivity to neutral solution components (osmolytes). The long term goals of the proposed studies are to investigate all the naturally-occurring solution factors that might influence RNA stability and identify the different strategies an RNA might use to accomplish a specific function in vivo. Specific questions will be investigated in each of three general areas: At a fundamental level, we will pursue our finding that some osmolytes stabilize RNA structures by affecting the hydration of RNA surfaces to obtain a more detailed picture of RNA hydration changes that accompany tertiary structure formation, and expand our studies of ions to include organic ions of in vivo relevance. Detailed studies will be made of the kinetics and energetics of conformational switches between alternative structures in two different RNAs in which the switch controls gene expression in response to changes in intracellular levels of free Mg2+ or a small metabolite (so-called "riboswitch" RNAs). These two RNAs use very different strategies to achieve stable tertiary structures, and probably function by different mechanisms as well; the studies are intended as explorations of these contrasting possibilities for functional RNAs. The physical studies of riboswitch RNAs will serve as a springboard for in vivo studies that will aim to elucidate the functional mechanism in the cellular environment, for example, how thermodynamic stability is balanced versus the kinetic rate of folding in determining functional response of RNA to cell signals, the reliance of RNAs on various ions found in vivo, and how changes in ion and osmolyte concentrations made by bacterial cells in response to changing growth conditions affect RNA function. These studies will help elucidate how RNAs function in a cellular environment, with potential applications in devising ways to block key RNA functions or design RNAs with specific regulatory functions. Bacterial pathogens are particularly reliant on riboswitch RNAs to survive in different environments inside and outside the human body, and are thus candidates for RNA-targeted therapeutics.