利用光鉗技術探討核醣體如何影響誘導框架位移的偽結

Abstract

-1計畫性核醣體框架位移 (-1 PRF)，對於細菌及病毒是很重要的轉譯機制，在相對較小的基因體中仍可以精確的調控不同蛋白質的比例。坐落於兩個開放閱讀框架之間的偽結或髮夾結構等mRNA的二級結構可以促進框架位移，此外，位於二級結構上游的滑動序列及中間的核苷酸數目也是影響-1 PRF發生的關鍵。-1 PRF的效率也和這些二級結構的穩定性、動態、plasticity (塑性)有高度的相關性。我們想要利用單分子光鉗的技術去研究這些會刺激-1 PRF的偽結結構與核醣體之間的交互作用，解釋-1 PRF的發生機制。 MMTV偽結是由小鼠乳腺腫瘤病毒而來的RNA 偽結，過去的研究指出它可以造成-1 PRF，然而，我們還不知道它的摺疊機制。另一個APK偽結則是由MMTV偽結突變而來的，但是它造成-1 PRF的效率(~2%) 卻比MMTV (~20%) 低許多，我們想要研究它們的摺疊機制以及它們與核醣體間的交互作用去釐清其中的原因。我們分別以30S小次單元以及70S核醣體結合於mRNA偽結結構的上游序列，結果發現這兩個偽結序列所摺疊而成的中間產物比核醣體結合前還少，且與30S結合後的比與70S結合後的更少一些，不過與70S結合的偽結結構穩定性稍微下降。我們推測核醣體會幫助偽結結構的摺疊，但是70S對於結構的影響更大。為了要摺疊成完整的結構，RNA必須和核醣體競爭回前幾個核苷酸，在此過程中可能會刺激-1 PRF的發生。 近幾年爆發的SARS與新冠肺炎(COVID-19)等嚴重的傳染病，分別是由SARS-CoV及 SARS-CoV-2所引起，並且這兩種病毒具有高度的基因相似性，甚至連它們會刺激-1 PRF的偽結結構也只有一個核苷酸之差而已，所造成-1 PRF的效率也相似 (~20%)，然而，我們並不太清楚這兩個偽結結構的摺疊機制。我們使核醣體占據mRNA偽結結構的上游序列，去影響偽結結構的摺疊動態，另外也以DNA handle甚至是突變的方式去觀察其改變。 我們推測核醣體會解開偽結中的上游結構，並且刺激下游序列的重新摺疊而促進整個偽結結構的形成，這些動態可能會促使-1 PRF的發生。

Minus-one programmed ribosomal frameshifting (-1 PRF) is an important translation mechanism for bacteria and viruses to generate accurate ratios of different proteins from their relatively small genomes. mRNA secondary structures such as pseudoknots (PKs) or hairpins located in the overlapping region between two open reading frames (OPFs) stimulate frameshifting. Besides, the slippery sequence and the spacer nucleotides are crucial to -1 PRF. The efficiency of -1 PRF is highly related to the stability, dynamics and the structural plasticity of these secondary structures. We aim to explore the interaction between the ribosome and -1 PRF-stimulating PKs to elucidate the frameshifting mechanism at the single molecule level by optical tweezers. The MMTV pseudoknot which was derived from the mouse mammary tumor virus has been studied previously for its role in -1 programmed ribosomal frameshifting. However, the detailed folding mechanism of MMTV is not well understood. APK is another pseudoknot derived from MMTV PK but its efficiency of -1 RPF (~2%) is lower than MMTV PK (~20%). Here, we bound the ribosomal 30S subunits or 70S ribosomes to the mRNA to study the effects of ribosomes on PK folding. In the presence of ribosomes, the intermediate formation ratio of these PK sequences is lower than the naked mRNA, and this ratio for the ribosomal 30S subunits is even lower than for the 70S ribosomes. It indicates that ribosomes stimulate the PK structure folding from the intermediate, and the 70S ribosomes is more influence to the structure. In order to maintain the stability of PK structures, nucleotides originally occupied by the ribosome will be retrieved to form the native PK and even to stimulate -1 PRF, but sometimes the mRNA fails to retrieve and folds intermediates. Some pandemic diseases outbroke in these years, including SARS and COVID-19 which are caused by SARS-CoV and SARS-CoV-2, respectively. The genomes of SARS-CoV-2 and SARS-CoV are highly similar and their frameshift-stimulating PKs even have only one nucleotide of difference. These two PKs have similar efficiencies of frameshifting (~20%). However, the detailed folding mechanism of these two PKs is not well understood. Therefore, we aim to investigate what factors could affect their folding, such as the bound ribosomes and masking the upstream sequence of PK by DNA handles. We bound the ribosome to the mRNA such that it would occupy some upstream nucleotides of PK to modulate the folding dynamics of the structure. We also mutated the corresponding nucleotides or cover them by DNA handles for comparison. We found that the folding pathway of PK was altered when its upstream sequence was covered by ribosomes or handles, or mutated. We hypothesize that the ribosome would unwind some upstream nucleotides of the PK structure or its folding intermediates during translation. Then, the downstream sequence is stimulated to refold in a different way, which in turn promotes the formation of the native PK structure. Thus, the nucleotides occupied by the ribosome will be retrieved to stimulate -1 PRF, alternatively, the folding dynamics may stimulate -1 PRF.