Single-molecule fluorescence resonance energy transfer studies of the human telomerase RNA pseudoknot: temperature-/urea-dependent folding kinetics and thermodynamics.
Single-molecule fluorescence resonance energy transfer studies of the human telomerase RNA pseudoknot: temperature-/urea-dependent folding kinetics and thermodynamics.
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DOI：
10.1021/jp501893c
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发表时间：
2014-04-10
期刊：
影响因子：
--
通讯作者：
Nesbitt DJ
中科院分区：
文献类型：
--
作者：
Holmstrom ED;Nesbitt DJ
核糖核蛋白端粒酶是一种RNA依赖的DNA聚合酶，催化一个短的，物种特异性的，DNA序列重复添加到线性真核细胞染色体的末端。端粒酶的单个RNA组分包含DNA合成的模板序列和功能关键的假结基序，其也可以作为不太稳定的发夹存在。在这里，我们使用最小版本的人端粒酶RNA假结研究这种发夹假结结构平衡，使用温度控制的单分子荧光共振能量转移（smFRET）实验。这些实验的尿素依赖性有助于确定折叠动力学和热力学。野生型的假结行为进行了比较和对比的突变假结序列牵连的遗传性疾病-先天性角化不良。这些发现清楚地表明，这种2nt非互补突变通过大幅降低折叠速率常数（约400倍）而仅名义上增加解折叠速率常数（约500倍）来使野生型假结的折叠不稳定。此外，尿素的依赖性的平衡和速率常数是用来开发一个自由能景观为这个单分子平衡，并提出有关过渡态的结构的细节。最后，尿素依赖的折叠实验提供了宝贵的物理见解的机制，不稳定的RNA假结的化学变性剂。
The ribonucleoprotein telomerase is an RNA-dependent DNA polymerase that catalyzes the repetitive addition of a short, species-specific, DNA sequence to the ends of linear eukaryotic chromosomes. The single RNA component of telomerase contains both the template sequence for DNA synthesis and a functionally critical pseudoknot motif, which can also exist as a less stable hairpin. Here we use a minimal version of the human telomerase RNA pseudoknot to study this hairpin–pseudoknot structural equilibrium using temperature-controlled single-molecule fluorescence resonance energy transfer (smFRET) experiments. The urea dependence of these experiments aids in determination of the folding kinetics and thermodynamics. The wild-type pseudoknot behavior is compared and contrasted to a mutant pseudoknot sequence implicated in a genetic disorder–dyskeratosis congenita. These findings clearly identify that this 2nt noncomplementary mutation destabilizes the folding of the wild-type pseudoknot by substantially reducing the folding rate constant (≈ 400-fold) while only nominally increasing the unfolding rate constant (≈ 5-fold). Furthermore, the urea dependence of the equilibrium and rate constants is used to develop a free energy landscape for this unimolecular equilibrium and propose details about the structure of the transition state. Finally, the urea-dependent folding experiments provide valuable physical insights into the mechanism for destabilization of RNA pseudoknots by such chemical denaturants.
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影响因子：
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