STRUCTURAL BASIS OF RNA THERMOSTABILITY

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在BioCAT进行的研究中，我们研究了结构和溶剂对RNA折叠和稳定性的贡献。两个问题指导了我们的小角X射线散射实验：1)RNA折叠中间体的动力学程度如何？2)什么相互作用负责维持中间构象？ 先前在BioCAT(2004)的实验表明，在0.4 mM的MgCl2背景下有一种中间的RNA构象。今年我们在不同浓度的单价阳离子背景下进行了SAXS实验。如果我们的RNA折叠中间体是高度动态的，我们预计单价阳离子会通过屏蔽由带负电荷的RNA骨架引起的静电斥力来增加这种动态性质。相反，我们发现这种RNA中间体在SAXS测量中即使在添加500 mM的氯化钠时也保持了恒定的大小和形状。这是一个非常令人惊讶的结果，表明中间构象不是非常动态的。此外，它表明静电斥力在维持这种构象中不起重要作用。 然后我们假设，该中间体核心的结构相互作用负责维持该中间体构象的奇异性质。我们进行了突变来破坏这些相互作用，然后访问了BioCAT进行后续实验。事实上，这种突变的RNA相对于野生型RNA是折叠的，尽管添加500 mM的氯化钠不会进一步折叠它。这些实验表明，这种独特的RNA折叠中间体是通过分子核心中的特定接触来稳定的。手稿正在准备中。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In research performed at BioCAT we have investigated structural and solvent contributions to RNA folding and stability. Two questions have guided our small-angle x-ray scattering experiments: 1) How dynamic are RNA folding intermediates? 2) What interactions are responsible for maintaining an intermediate conformation? Previous experiments at BioCAT (2004) indicated an intermediate RNA conformation in the background of 0.4 mM MgCl_2 . This year we performed SAXS experiments in the background of various concentrations of monovalent cations. If our RNA folding intermediate is highly dynamic we would anticipate monovalent cations to increase this dynamic nature through shielding of the electrostatic repulsion caused by the negatively charged RNA backbone. Instead we found that this RNA intermediate maintained a constant size and shape as measured by SAXS even upon addition of 500 mM NaCl. This was a very surprising result and suggests that the intermediate conformation is not very dynamic. Additionally, it suggests that electrostatic repulsion does not play a significant role in maintaining this conformation. We then hypothesized that structural interactions in the core of this intermediate were responsible for maintaining the singular nature of this intermediate conformation. We made mutations to disrupt these interactions and then visited BioCAT for follow-up experiments. Indeed this mutated RNA is collapsed relative to the wild-type RNA though the addition of 500 mM NaCl does not collapse it further. These experiments indicate that this distinct RNA folding intermediate is stabilized by particular contacts in the core of the molecule. Manuscript in preparation.