Melnykov Artem V, Nayak Rajesh K, Hall Kathleen B, Van Orden Alan
†Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, United States.
‡Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523, United States.
Biochemistry. 2015 Mar 17;54(10):1886-96. doi: 10.1021/bi5014276. Epub 2015 Mar 4.
RNA hairpins are ubiquitous structural elements in biological RNAs, where they have the potential to regulate RNA folding and interactions with other molecules. There are established methods for predicting the thermodynamic stability of an RNA hairpin, but there are still relatively few detailed examinations of the kinetics of folding. Nonetheless, several recent studies indicate that hairpin folding does not proceed via a simple two-state model. Here, we monitor fluorescence from hairpins constructed as molecular beacons in ensemble, fluorescence correlation spectroscopy, and stopped-flow experiments to describe the folding of RNA hairpins with long (15 nucleotide) loops. Our results show that folding of these hairpins occurs through more than two states and that the mechanism of folding includes a fast intermediate phase observed on the tens of microseconds time scale and a slow phase, attributed to formation of the native folded hairpin loop and stem, observed on the milliseconds time scale. The composition of the RNA loop determines the time scale of intermediate and native folded states. Hairpins with a polyuracil loop sequence exhibit slower relaxation of the intermediate state and faster relaxation of the native folded state when compared to that of hairpins with cytosine or adenine in the loop. We hypothesize this composition dependence could be attributed to nucleobase stacking in cytosine and adenine containing regions of the loop, which would be absent in hairpins containing polyuracil loops. Such base stacking could destabilize the intermediate folds, thereby speeding the relaxation of the intermediate relative to similar sized hairpins with no base stacking in the loop. Likewise, the lower intermediate stability could prolong the relaxation of the native folded state.
RNA发夹结构是生物RNA中普遍存在的结构元件，它们有可能调节RNA折叠以及与其他分子的相互作用。目前已有预测RNA发夹结构热力学稳定性的方法，但对折叠动力学的详细研究仍然相对较少。尽管如此，最近的几项研究表明，发夹折叠并非通过简单的两态模型进行。在这里，我们通过监测作为分子信标构建的发夹在整体实验、荧光相关光谱和停流实验中的荧光，来描述具有长（15个核苷酸）环的RNA发夹的折叠过程。我们的结果表明，这些发夹的折叠通过两个以上的状态发生，并且折叠机制包括在几十微秒时间尺度上观察到的快速中间相和在毫秒时间尺度上观察到的缓慢相，后者归因于天然折叠发夹环和茎的形成。RNA环的组成决定了中间态和天然折叠态的时间尺度。与环中含有胞嘧啶或腺嘌呤的发夹相比，具有聚尿嘧啶环序列的发夹中间态的弛豫较慢，而天然折叠态的弛豫较快。我们推测这种组成依赖性可能归因于环中含胞嘧啶和腺嘌呤区域的碱基堆积，而含有聚尿嘧啶环的发夹中不存在这种堆积。这种碱基堆积可能会使中间折叠不稳定，从而相对于环中无碱基堆积的类似大小的发夹，加速中间态的弛豫。同样，较低的中间稳定性可能会延长天然折叠态的弛豫时间。
