以單分子螢光共振能量轉移技術探討SARS-CoV-2引起框架轉移偽結在轉譯過程中可能形成之中間構形

Abstract

許多細菌或病毒(例如SARS-CoV-2)利用-1 Programmed Ribosomal Frameshifting (-1PRF)的發生來改變開放閱讀框，進而重新排列下游密碼子以產生不同胺基酸。目前尚不清楚SARS-CoV-2 RNA在轉譯的過程中是否形成中間結構以及這些結構如何影響其-1PRF的效率，因此我們利用single-molecule FRET (smFRET)來觀察SARS-CoV-2 RNA可能形成的中間構形。透過strand displacement策略模擬mRNA在轉譯過程中結構依序由上游重新摺疊的情況，其中一種設計為將螢光分子標定在DNA handle上(CoV2PK74)，另一種設計為直接在RNA上標定螢光分子，以去除額外DNA handle可能造成的影響。我們發現經過重新摺疊後的CoV2PK74中在FRET族群分布圖中出現新的族群，說明重新摺疊後的RNA可能形成中間構形。另外在螢光標定的RNA實驗中，我們發現有stem 2不穩定之中間構形。通過干擾stem 1並觀察結構穩定性，我們發現到中間構形的減少，說明干擾stem 1會誘導中間構形重新排列形成更穩定的偽結。 根據這些實驗結果，我們推論在SARS-CoV-2 RNA偽結的重新摺疊過程中會出現中間構形，而由上游逐漸接近的核醣體會干擾5’端stem 1之結構使RNA重新排列成穩定的偽結，最後造成閱讀框架的位移。

Many bacteria or viruses (such as SARS-CoV-2) rely on the occurrence of -1 Programmed Ribosomal Frameshifting (-1 PRF) events to shift the open reading frame, leading to the rearrangement of downstream codons necessary for producing the proteins of different sequences. Whether SARS-CoV-2 RNA forms intermediate structures and how they affect its -1 PRF remains unknown. We used single-molecule FRET to probe the potential structures formed by SARS-CoV-2 RNA. Using the strand displacement strategy, we can mimic the vectorial folding of mRNA strcutures during translation. One construct was labeled with dyes on the DNA handles (CoV2PK74) and the other constructs were directly labeled with dyes on the RNA to minimize the potential influence of artificial handles. We found that the FRET distribution in CoV2PK74 shows more than one population, suggesting there are intermediates. In dye-labeled RNA, we found intermediates with unstable structures in stem 2. By partially disrupting the upstream stem 1, we observed a reduction in the intermediate population, suggesting that the intermediates rearrange into a more stable pseudoknot. Based on these experimental results, we propose that conformational intermediates are generated during the refolding process of SARS-CoV-2 RNA, and perturbation of the 5' structural domain by the translating ribosome allows the RNA to rearrange into a stable pseudoknot, resulting in -1 ribosomal frameshifting.