利用單分子技術光鉗探討人類端粒酶RNA偽結結構的摺疊機制

Abstract

病毒經常利用-1核醣體框架位移來表現特定的結構蛋白及酵素(例如：HIV, SARS-CoV 及IBV)，當核醣體在轉譯mRNA成蛋白質時，mRNA上的偽結結構會刺激框架位移，而且偽結結構穩定性與框架位移之效率呈現正相關。 DU177此偽結結構是由人類端粒酶RNA修改而來，我們利用DU177當model進行實驗操作，此結構包含兩個stem及兩個loop，且為重疊性的hairpin構成，而其中stem與loop之間有些核苷酸有Hoogsteen base pair的形成，並且產生5組三重鹼基對，進而增強了此三級結構的穩定性。 本研究利用單分子技術，以光鉗對DU177施加外力，並且測量解開此結構所需之外力與打開的長度，進而推導其原先的結構為何。此外，我們也建構不同DU177的突變序列和結構，進行實驗操作，來推測DU177的摺疊機制。我們在實驗中發現，大部分的DU177會形成hairpin或是一些較不穩定的intermediate結構，僅六分之一比例的DU177會形成完整穩定的偽結結構。在綜合一系列的實驗結果後，我們推論形成偽結結構有兩種主要途徑，第一種是先形成hairpin 1並且以此結構穩定存在著，且較不易進一步形成完整穩定的偽結結構；另一種為hairpin 2先形成，且hairpin 2一旦形成，即可迅速形成帶有三重鹼基的穩定偽結結構。

Viruses often use -1 ribosomal frameshifting to express specific enzymes and structural proteins (e.g. HIV, SARS-CoV and IBV). When the ribosome translates an mRNA, a pseudoknot structure on the mRNA can stimulate frameshifting, and the efficiency is positively correlated with its mechanical strength, instead of the thermodynamic stability. Here we used the DU177 RNA pseudoknot, which is modified from the human telomerase RNA, as a model system to study the folding and unfolding mechanisms of a typical frameshift-stimulating RNA pseudoknot. DU177 consists of two overlapping hairpins, with five base triples in between that give extra stability to the whole structure. We used optical tweezers to measure and analyze the forces and extensions of a series of DU177 mutants during unfolding and refolding. We found that in most cases DU177 folds into pseudoknot-like intermediate structures or hairpins alone. Only one sixth of the RNA molecules completely refold to the native structure from the fully unfolded state. With a series of related experiments, we propose a folding pathway of the pseudoknot, where DU177 is usually trapped in a stable hairpin-1-containing intermediate and rarely proceeds to form the native pseudoknot. On the other hand, if the less stable hairpin 2 is formed first, refolding toward the native state is favored, as no stable intermediates were detected from the experiments.