Triplex structures in an RNA pseudoknot enhance mechanical stability and increase efficiency of-1 ribosomal frameshifting
Triplex structures in an RNA pseudoknot enhance mechanical stability and increase efficiency of-1 ribosomal frameshifting
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DOI：
10.1073/pnas.0905046106
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发表时间：
2009-08-04
影响因子：
11.1
通讯作者：
Tinoco, Ignacio, Jr.
中科院分区：
文献类型：
--
作者：
Chen, Gang;Chang, Kung-Yao;Tinoco, Ignacio, Jr.
许多病毒使用程序化的-1核糖体移码来表达特定比例的结构蛋白和酶蛋白。信使RNA中的假结结构刺激上游光滑序列中的帧移位。假结机械稳定性和移码效率的详细分子决定因素还不是很清楚。在这里，我们使用光钳的单分子展开研究和移帧试验来阐明假结的机械稳定性和移帧效率是如何受到三级茎环相互作用的调节的。对一个模型假结和旨在剖析特定相互作用的突变体的机械展开表明，机械稳定性强烈依赖于茎-环相互作用形成的三链结构。结合单分子和突变研究有助于假结折叠中间体的鉴定。伪结和突变体的平均展开力在50到22皮牛顿之间，与移帧效率从53%到0%相关。大槽和小槽三股结构的形成提高了假节茎的稳定性和抗扭力，从而可能刺激帧移位。更好地了解移码效率的分子决定因素可能有助于针对移码的抗病毒疗法的发展。
Many viruses use programmed -1 ribosomal frameshifting to express defined ratios of structural and enzymatic proteins. Pseudoknot structures in messenger RNAs stimulate frameshifting in upstream slippery sequences. The detailed molecular determinants of pseudoknot mechanical stability and frameshifting efficiency are not well understood. Here we use single-molecule unfolding studies by optical tweezers, and frameshifting assays to elucidate how mechanical stability of a pseudoknot and its frameshifting efficiency are regulated by tertiary stem-loop interactions. Mechanical unfolding of a model pseudoknot and mutants designed to dissect specific interactions reveals that mechanical stability depends strongly on triplex structures formed by stem-loop interactions. Combining single-molecule and mutational studies facilitates the identification of pseudoknot folding intermediates. Average unfolding forces of the pseudoknot and mutants ranging from 50 to 22 picoNewtons correlated with frameshifting efficiencies ranging from 53% to 0%. Formation of major-groove and minor-groove triplex structures enhances pseudoknot stem stability and torsional resistance, and may thereby stimulate frameshifting. Better understanding of the molecular determinants of frameshifting efficiency may facilitate the development of anti-virus therapeutics targeting frameshifting.