Effect of loop composition on the stability and folding kinetics of RNA hairpins with large loops.
Effect of loop composition on the stability and folding kinetics of RNA hairpins with large loops.
复制标题
环组成对大环 RNA 发夹稳定性和折叠动力学的影响。
DOI：
10.1021/bi5014276
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发表时间：
2015
期刊：
影响因子：
2.9
通讯作者：
VanOrden,Alan
中科院分区：
文献类型：
--
作者：
Melnykov,ArtemV;Nayak,RajeshK;Hall,KathleenB;VanOrden,Alan
RNA发夹是生物RNA中普遍存在的结构元件，它们具有调节RNA折叠和与其他分子相互作用的潜力。已经有了预测RNA发夹热力学稳定性的既定方法，但对折叠动力学的详细检查仍然相对较少。尽管如此，最近的几项研究表明，发夹折叠不是通过简单的两态模型进行的。在这里，我们监测作为分子信标的发夹的荧光，荧光相关光谱和停流实验，以描述RNA发夹的折叠与长(15个核苷酸)环。我们的结果表明，这些发夹的折叠发生在两个以上的状态，折叠的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尺度上观察到的慢相，归因于自然折叠发夹环和发夹茎的形成。RNA环的组成决定了中间折叠状态和自然折叠状态的时间尺度。与环中含有胞嘧啶或腺嘌呤的发夹相比，具有多尿嘧啶环序列的发夹表现出中间状态的较慢松弛和自然折叠状态的较快松弛。我们假设这种成分依赖性可能归因于核碱基在环的胞嘧啶和腺嘌呤含有区域的堆积，这在含有多尿嘧啶环的发夹中是不存在的。这种碱基堆积可能会破坏中间折叠的稳定性，从而相对于环中没有碱基堆叠的类似尺寸的发夹加快中间折叠的松弛。同样，较低的中间稳定性可以延长自然折叠状态的松弛。
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.