Factors stabilizing RNA structures

描述(申请人提供)：许多RNA的生物学功能依赖于紧密的三级结构，具有不规则的骨架构象和紧密的磷酸盐并列。由此产生的不利的静电自由能是折叠的主要障碍，并导致RNA三级结构比二级结构对离子和溶剂环境更敏感。这些研究的长期目标是提供一幅离子和溶剂相互作用稳定RNA三级结构的系统和定量的图像，并将该图像与RNA的潜在静电性质联系起来。这些研究的一个主要目的是获得一系列RNA结构的准确的镁2-RNA结合等温线，这些结构展示了所有潜在的镁结合环境，包括完全由完全水合的镁稳定的RNA，其中镁(H20)6[2]可能与RNA结构中的口袋或通道氢键结合的RNA，在特定位置结合了高度脱水离子的RNA，以及模拟大RNA(如核糖体)中螺旋紧密堆积的RNA。还将研究镁与部分折叠状态(仅二级结构)捕获的RNA的结合，以提供折叠反应过程中镁结合的净变化图。新开发的基于荧光的直接测量游离和RNA结合的镁的方法将被用于这些研究。其他研究将考察所提出的“静电崩塌”现象，将其作为大RNA折叠过程中一种独立的、依赖于镁离子的转变，着眼于RNA对单价离子的选择性，这可能是紧凑RNA的一个普遍特征，反映了靠近RNA表面的单价离子的部分脱水，以及可用作离子探针的小肽或蛋白质的结合，以及稳定RNA结构的水合水的结合。最后，这些结果将被用作与离子-RNA相互作用的理论预测进行比较的基准，特别是最近开发的一个框架，该框架使用非线性Poisson-Boltzmann方程来描述水合RNA结合的Mg2，以及一个Born模型来计算RNA中特定位置部分脱水离子的能量学。预测RNA三级构象的依赖于镁的折叠自由能的能力在许多情况下都是有用的。

DESCRIPTION (provided by applicant): The biological functions of many RNAs depend on compact tertiary structures with irregular backbone conformations and close juxtapositions of phosphates. The resulting unfavorable electrostatic free energy is a major barrier to folding, and causes RNA tertiary structure to be much more sensitive to the ion and solvent environment than is secondary structure. The long term goal of these studies is to provide a systematic and quantitative picture of ion and solvent interactions stabilizing RNA tertiary structures, and relate the picture to the underlying electrostatic properties of RNA. A major aim of the proposed studies is to obtain accurate Mg 2+ - RNA binding isotherms for a set of RNA structures that illustrate the full range of potential Mg 2+ binding environments, including RNAs stabilized entirely by fully hydrated Mg 2+, RNAs in which Mg(H20)6[2]+ may be hydrogen bonded to pockets or channels in the RNA structure, RNAs with highly dehydrated ions bound atspecific sites, and RNAs that mimic the close packing of helices in large RNAs such as the ribosome. Mg 2+ binding to RNAs trapped in partially folded states (secondary structure only) will also be studied, to provide a picture of the net change in Mg 2+ binding during the folding reaction. Newly developed fluorescence-based methods for directly measuring free and RNA-bound Mg 2+ will be used in these studies. Other studies will look at the proposed phenomenon of "electrostatic collapse" as a separate, Mg 2+- dependent transition in folding of large RNAs, at the selectivity of RNAs for monovatent ions, which may be a widespread feature of compact RNAs and reflect partial dehydration of monovalent ions bound close to the RNA surface, and at binding of small peptides or proteins that may serve as probes of ions and hydrating water stabilizing an RNA structure. Lastly, these results will be used as benchmarks for comparison with theoretical predictions of ion - RNA interactions, particularly a recently developed framework that uses the non-linear Poisson-Boltzmann equation to describe hydrated RNA-bound Mg 2+ and a Born model to compute the energetics of partially dehydrated ions at specific locations in the RNA. The ability to predict the Mg2+-dependent folding free energies of RNA tertiary conformations would be useful in a number of contexts.