STRUCTURAL BASIS OF RNA THERMOSTABILITY

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 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折叠中间体的动态如何？2)什么样的相互作用负责维持中间构象？ 先前在BioCAT（2004）的实验表明在0.4mM MgCl_2的背景下的中间RNA构象。今年，我们在各种浓度的一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，我们将预期单价阳离子通过屏蔽由带负电荷的RNA骨架引起的静电排斥来增加这种动态性质。相反，我们发现这种RNA中间体保持恒定的大小和形状，如通过SAXS测量的，即使在添加500 mM NaCl后。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，这表明静电排斥在维持这种构象中不起重要作用。 然后，我们假设，在这个中间体的核心结构的相互作用是负责保持这个中间体构象的奇异性。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是塌陷的，尽管添加500 mM NaCl不会使其进一步塌陷。这些实验表明，这种独特的RNA折叠中间体通过分子核心中的特定接触而稳定。 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.