Swisher Jennifer F, Su Linhui J, Brenowitz Michael, Anderson Vernon E, Pyle Anna Marie
Integrated Program in Cellular, Molecular, and Biophysical Studies, Columbia University, New York, NY 10032, USA.
J Mol Biol. 2002 Jan 18;315(3):297-310. doi: 10.1006/jmbi.2001.5233.
Group II introns are large catalytic RNA molecules that fold into compact structures essential for the catalysis of splicing and intron mobility reactions. Despite a growing body of information on the folded state of group II introns at equilibrium, there is currently no information on the folding pathway and little information on the ionic requirements for folding. Folding isotherms were determined by hydroxyl radical footprinting for the 32 individual protections that are distributed throughout a group II intron ribozyme derived from intron ai5gamma. The isotherms span a similar range of Mg(2+) concentrations and share a similar index of cooperativity. Time-resolved hydroxyl radical footprinting studies show that all regions of the ribozyme fold slowly and with remarkable synchrony into a single catalytically active structure at a rate comparable to those of other ribozymes studied thus far. The rate constants for the formation of tertiary contacts and recovery of catalytic activity are identical within experimental error. Catalytic activity analyses in the presence of urea provide no evidence that the slow folding of the ai5gamma intron is attributable to the presence of unproductive kinetic traps along the folding pathway. Taken together, the data suggest that the rate-limiting step for folding of group II intron ai5gamma occurs early along the reaction pathway. We propose that this behavior resembles protein folding that is limited in rate by high contact order, or the need to form key tertiary interactions from partners that are located far apart in the primary or secondary structure.
II类内含子是大型催化RNA分子，可折叠成紧密结构，这对剪接催化和内含子移动反应至关重要。尽管关于II类内含子在平衡状态下折叠状态的信息越来越多，但目前尚无关于折叠途径的信息，关于折叠的离子需求的信息也很少。通过羟基自由基足迹法测定了来自内含子ai5γ的II类内含子核酶中分布的32个单独保护区域的折叠等温线。这些等温线跨越了相似的Mg(2+)浓度范围，并具有相似的协同指数。时间分辨羟基自由基足迹研究表明，核酶的所有区域折叠缓慢，且以与迄今为止研究的其他核酶相当的速率显著同步地折叠成单一的催化活性结构。在实验误差范围内，三级接触形成和催化活性恢复的速率常数相同。在尿素存在下的催化活性分析没有提供证据表明ai5γ内含子的缓慢折叠归因于折叠途径中存在非生产性动力学陷阱。综合来看，数据表明II类内含子ai5γ折叠的限速步骤发生在反应途径的早期。我们提出，这种行为类似于蛋白质折叠，其速率受高接触序限制，或者需要从一级或二级结构中相距很远的伙伴形成关键的三级相互作用。
