KINETICS AND THERMODYAMIC RNA FOLDING INTERMEDIATES MONITORED BY SAXS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。RNA，曾经被认为主要是作为遗传信息的信使，现在被认为在剪接体和核糖体等复合体中起着核心的结构和酶的作用。为了发挥作用，RNA必须折叠成一个离散的三维结构，这就引入了RNA折叠问题。我们之前的SAXS工作表明，四膜虫I群核酶在折叠过程中经历了一个大的，依赖于Mg2+的折叠状态，从变性状态到折叠状态，这种崩溃发生的速度远远快于整体折叠。此外，我们还发现，在观察到任何第三纪构造形成之前，坍塌基本上已经完成，这表明在褶皱的早期就存在一种非特异性的坍塌状态。在这里，我们建议在这些观察的基础上，通过(i)获得关于崩塌状态形成后填充的中间体进展的结构信息；（ii）探测影响塌缩态性质的分子特征；（iii）监测核酶被尿素变性后填充的中间体的整体结构。这些实验是一个更大项目的一部分，该项目旨在描述这种RNA的折叠行为和整体折叠情况。

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. RNA, once thought to serve mainly as a courier of genetic information, is now known to play central structural and enzymatic roles in such complexes as the spliceosome and ribosome. To function, RNA must fold to a discrete three-dimensional structure, which introduces the RNA folding problem. Our previous SAXS work demonstrated that the Tetrahymena group I ribozyme undergoes a large, Mg2+-dependent collapse in folding from the denatured state to the folded state, and that collapse occurs substantially faster than overall folding. Further, we found that collapse is largely complete before any tertiary structure is observed to form, suggesting that a non-specifically collapsed state is populated early in folding. Here we propose to build on these observations by (i) obtaining structural information on the progression of intermediates that is populated after formation of the collapsed state; (ii) probing molecular features that affect the properties of the collapsed state; and (iii) monitoring the global structures of intermediates that are populated as the ribozyme is denatured by urea. These experiments are part of a larger project aimed at characterizing the folding behavior and the overall folding landscape for this RNA.