以單分子螢光共振能量轉移觀測mRNA的長度對反義寡核苷酸以及30S核醣體次單元的交互作用影響

Abstract

在細菌的轉譯過程中，存在多個與mRNA結構相關的因素參與調控。例如，轉譯起始序列位於mRNA起始密碼子附近，提供核醣體結合位置，其結構和序列特徵影響轉譯的起始效率。轉譯起始序列包括Shine-Dalgarno序列（SD序列），它能與核醣體結合，幫助定位正確的起始密碼子。此外，細菌mRNA的非編碼區域（5' UTR和3' UTR）的一些特性亦可能具有調控轉譯的潛力，包含序列、結構和轉譯調控蛋白質(或核醣體)的結合位點。在本篇論文，我們主要利用單分子螢光共振能量轉移（smFRET）技術，觀察rpsO基因不同長度的mRNA與反義寡核苷酸 (antisense oligomers) 和30S核醣體次單元的相互作用。我們利用反義寡核苷酸與mRNA的黏合模擬30S次單元的結合，結果發現黏合的效率普遍偏低，這同時也說明了30S上其他核醣體蛋白與核醣體RNA的重要性。接下來的實驗中，觀察到標記螢光的30S核醣體次單元與三種不同長度的mRNA進行作用時，發現具有完整上下游序列的mRNA與30S核醣體次單元之間的動態結合最為明顯，這說明mRNA的長度與30S核醣體次單元的結合有高度相關的關聯性，可能和核醣體搜尋mRNA上面的轉譯起始位有關。

Multiple factors related to mRNA structures are involved in the regulation of translation in bacteria. For instance, the translation initiation site, located near the mRNA start codon, provides a binding site for ribosomes, and its structural and sequence features influence the efficiency of translation initiation. The translation initiation site includes the Shine-Dalgarno (SD) sequence, which binds to the ribosome and helps to position the correct start codon. Translation initiation factors, such as IF1, IF2, and IF3, regulate the initiation process in bacteria. Additionally, the non-coding regions of bacterial mRNA, namely the 5' untranslated region (5' UTR) and 3' UTR, have the potential to regulate translation. These regions may contain regulatory sequences, structural elements, and binding sites for translational regulatory proteins and even ribosomes. In this study, we primarily employ the single-molecule fluorescence resonance energy transfer (smFRET) technique to observe the interaction between antisense oligomers, as well as the 30S ribosomal subunit, and the mRNA of the rpsO gene with varying lengths. We anneal antisense oligomers to mRNA to mimic the binding of the 30S ribosomal subunit. The results showed that the efficiency is generally low, suggesting the importance of ribosomal proteins and ribosomal RNA on the 30S subunit. However, observations of fluorescence-labeled 30S subunits interacting with mRNAs of different lengths indicated that mRNAs containing the complete downstream coding sequence exhibited the most dynamic binding with the 30S subunit. These preliminary data suggest a significant correlation between the length of mRNA and the 30S ribosomal subunit, which may be related to the searching mechanism of 30S subunits for the translation initiation site.