使用單分子螢光共振能量轉移技術研究螢光標記之核醣體亞基如何搜尋訊息核醣核酸的轉譯起始位

Using Fluorescence-labeled 30S Ribosomal Subunits to Study How They Find mRNA Translation Initiation Sites by Single-molecule FRET

廖仁豪(Ren-Hao Liao)

指導教授 ： 溫進德

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202101906

摘要

細菌中mRNA的轉譯效率顯著受到起始階段的影響。在起始階段，核醣體的30S次單元(亞基)與mRNA的結合，主要是透過位於mRNA起始密碼子上游約6 個鹼基的Shine-Dalgarno (SD) 與30S次單元16S rRNA中的anti-SD (aSD) 序列互補。先前研究表明，SD-aSD 相互作用穩定了30S-mRNA的複合體，並在起始因子 (initiation factor) 和起始tRNA協助下，30S次單元固定在起始密碼子處。然而，關於mRNA 如何招募30S次單元以及30S次單元如何搜索SD序列，得知甚少。在本論文中，我們通過單分子螢光共振能量轉移 (smFRET) 觀察了螢光標記的30S次單元與mRNA的相互作用。我們的觀察表明，30S 亞基首先與位於 5’-非轉譯區 (5’-UTR) 的非特異性位點結合，然後通過未知機制轉移到起始位點。此外，具有長且非結構化 5’-UTR 的人工mRNA (GAA52-sRBS)具有高30S次單元招募效率，但此效率是否與自然發生的mRNA類似則是未知。因此，我們透過和E. coli 中具有高轉譯效率的lpp mRNA進行比較，發現GAA52-sRBS的招募效率為lpp mRNA的11倍。然而，GAA52-sRBS 的實際轉譯效率僅為lpp的50%。此外，透過smFRET的即時觀察，發現30S次單元可能會與lpp 5’-UTR互動。這些結果說明轉譯起始的機制比我們的預期複雜。在後續的實驗中，我們將利用新的螢光標記30S次單元，將標記置於靠近mRNA5’端出口處的S6蛋白，此類30S次單元將可偵測5’-UTR的動態變化，以揭示更多轉譯起始機制的細節。

關鍵字

轉譯起始 ； 30S次單元(亞基) ； 核醣體 ； mRNA招募 ； 單分子螢光共振能量轉移

並列摘要

Translation efficiency of mRNA in bacteria is significantly influenced by the initiation phase. During the initiation stage, the ribosomal 30S subunit binds to the mRNA through the base pairing between the AG-rich Shine-Dalgarno (SD) sequence, which is located at about 6 nucleotides upstream of the start codon on the mRNA, and the anti-SD (aSD) on the 16S rRNA of the 30S subunit. The SD-aSD interaction stabilizes the complex of 30S-mRNA and helps the 30S subunit locate at the start codon in the presence of initiation factors and initiator tRNA. However, little is known about how the mRNA recruits the 30S subunit and how the 30S subunit searches for the SD sequence. This study is to observe the interaction between the fluorescence-labeled 30S subunits and mRNA by single-molecule Fluorescence Resonance Energy Transfer (smFRET). Our observation indicates that the 30S subunit binds to nonspecific sites locating at the 5’-end untranslated region (5’-UTR) first and transfers to the initiation sites through an unknown mechanism. Moreover, a synthetic mRNA with long and unstructured 5’-UTR (GAA52-sRBS) shows a high 30S recruiting efficiency (RE). However, this efficiency is not biologically relevant. The natural mRNA lpp, which shows high translation efficiency in E. coli, is adopted to evaluate the RE of GAA52-sRBS. We find that the RE of GAA52-sRBS is 11 times higher than lpp mRNA. However, the measured translation efficiency of GAA52-sRBS is only ~ 50% of lpp mRNA. The real-time observation of the interaction between the 30S subunit and lpp 5’-UTR by smFRET indicates that the 30S may locate at 5’-UTR first. These results suggest that the mechanism of 30S recruitment for mRNA is more complicated than what we have expected. In the future, a new fluorescence-labeled 30S subunit at S6 protein, where is close to the 5’ exit site of mRNA, will be used to reveal the detail of the 30S interacting with 5’-UTR of mRNA in initiation.

並列關鍵字

Translation initiation ； 30S subunit ； ribosome ； mRNA recruitment ； single-molecule Fluorescence Resonance Energy Transfer

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