TIME RESOLVED HYDROXYL RADICAL FOOTPRINTING OF RNA

大的催化RNA和RNA加工复合体的构象动态变化是其功能所固有的。尽管对tRNA的实验表明，RNA三级结构可以在不到一秒的时间内形成，但很少有实验方法能够在这个时间尺度上以高分辨率探测大的RNA结构。一种新开发的使用同步加速器x射线束的停流羟基自由基裂解RNA的方法可以在20-30毫秒内绘制出RNA的溶剂可及表面。将这种方法应用于四膜虫核酶的折叠，结果表明，在10 mM的氯化镁存在下，最稳定的结构域P4-P6以1s-1的速度形成，而其他结构域需要几分钟才能完全折叠。X射线羟基自由基足迹将被用来研究RNA三级折叠的基本机制。了解RNA结构是如何形成的，将有助于更好地预测自然序列和设计序列的生物学功能。四膜虫核酶是一个理想的模型体系，因为天然结构的形成与RNA的催化活性密切相关。核酶的独立折叠的P4-P6结构域将用于研究一簇紧密结合的镁离子对三级相互作用的成核作用。环境因素，如温度和离子强度，对折叠动力学的影响将被确定。该折叠机制将与来自Azoarcus sp.的一个小的单结构域I内含子的折叠机制进行比较。新合成的核酶RNA的折叠路径将与全长转录本的折叠进行比较。探测毫秒级时间尺度上的三级相互作用的能力将使研究底物诱导的折叠核酶的构象变化成为可能。将使用模拟5‘和3’剪接位点的RNA底物来研究剪接第一步和第二步之间的活性位点重排。在生理条件下，剪接过程中的构象变化被认为是在几秒钟内发生的，非常适合于通过停流X射线足迹进行研究。拟议的实验将为RNA加工复合体中的底物识别和校对机制提供见解。

Dynamic change in the conformation of large catalytic RNAs and RNA- processing complexes is intrinsic to their function. Although experiments on tRNA suggest that RNA tertiary structures can form in less than one second, few experimental methods are capable of probing large RNA structures with high resolution on this timescale. A newly developed method for stopped-flow hydroxyl radical cleavage of RNA using a synchrotron x-ray beam can map the solvent-accessible surface of an RNA in 20-30 milliseconds. Application of this method to folding of the Tetrahymena ribozyme showed that the most stable domain, P4-P6, forms at a rate of 1s-1 in the presence of 10 mM MgC12, whereas other domains require minutes to be completely folded. X-ray hydroxyl radical footprinting will be used to investigate fundamental mechanisms of tertiary folding in RNA. An understanding of how RNA structures are formed will lead to better predictions of biological function for both natural and designed sequences. The Tetrahymena ribozyme is an ideal model system, because the formation of native structure is correlated with the well-studied catalytic activity of the RNA. The independently folding P4-P6 domain of the ribozyme will be used to investigate nucleation of tertiary interactions by a cluster of tightly bound magnesium ions. The effect of environmental factors, such as temperature and ionic strength, on the folding kinetics will be determined. The folding mechanism will be compared to that of a small, single-domain group I intron from Azoarcus sp. The folding pathway of newly synthesized ribozyme RNA will be compared to refolding of full- length transcripts. The ability to probe tertiary interactions on the millisecond timescale will make it possible to investigate substrate- induced conformational changes in the folded ribozyme. Active site rearrangements between the first and second step of splicing will be studied using RNA substrates that mimic the 5' and 3' splice sites. Conformational changes during splicing are thought to occur in seconds under physiological conditions, and are ideally suited to investigation by stopped-flow x-ray footprinting. The proposed experiments will provide insights into substrate recognition and proofreading mechanisms in RNA-processing complexes.