藉由細胞外轉譯方式研究核醣體蛋白bS1對mRNA 5’端非轉譯區的調控表現

Abstract

bS1是細菌中分子量最大的核醣體蛋白，像一座橋作用於mRNA以及核醣體之間，並且具有解開較弱的mRNA二級結構的能力。有些具有結構的mRNA是需要bS1才能有效進行轉譯。此外bS1對於那些因缺少Shine-Dalgarno序列而與核醣體結合力較弱的mRNA是必不可少的。本研究中，我們藉由細胞外轉譯以及luciferase assay的實驗發現bS1會影響具有不同5’端非轉譯區(5’UTR)的mRNA轉譯。我們挑選4種mRNA 5’UTR，分別是bS1自身基因且5’UTR具有3個髮夾結構的rpsA、uS15自身基因且具有兩個髮結結構並可以自動轉換成偽結的rpsO、二級結構強度較弱的ET24以及lpp。我們發現bS1可以抑制rpsA的轉譯效率，而此效果與rpsA的結構以及bS1的功能和與核醣體的作用具有相關性。bS1對rpsO的轉譯效率不具有明顯的影響。對ET24以及lpp的轉譯皆具有促進效果，只是隨著bS1濃度提高，ET24促進的效果降低，lpp則是不受影響。另外我們也發現bS1的mutant也會造成不同的影響。相較於bS1-His6(C端有His tag)可以有效抑制rpsA轉譯效率，bS1-dD6(去除domain 6)則可以促進。然而bS1-His6以及bS1-dD6皆是促進rpsO的轉譯效率。我們的結果顯示，bS1和mRNA 5’UTR之間的交互作用可以影響轉譯效率，但其中的機制還未明朗，需要更深入的探討。

bS1 is the largest ribosomal protein in bacteria. It acts like a bridge between messenger RNA (mRNA) and the ribosome and has the ability to unfold weak secondary structures of the mRNA. Some mRNAs with structures require bS1 for efficient translation. In addition, bS1 is indispensable for mRNA which lacks the Shine-Dalgarno sequence, such that the ribosome binds weakly to it. In our study, we investigate how bS1 affects translation of mRNA with different 5’ untranslated region (5’ UTR) by in vitro translation, followed by renilla luciferase assay. Four 5’ UTRs are chosen in this study: rpsA, which encodes the bS1, has three hairpin structures; rpsO, which encodes the uS15, has two hairpin structures that can be spontaneously converted to a pseudoknot; ET24 and lpp are single-stranded RNA with weak structures. We observed that bS1 could inhibit the translation of rpsA; this inhibition is dependent on the rpsA structure and correlated with the bS1 function and its interaction with the ribosome. bS1 did not apparently affect the translation of rpsO, whereas bS1 promoted translation of ET24 and lpp. The level of promotion for ET24, but not for lpp, decreased with increasing bS1 concentration. Additionally, we also found that bS1 mutants could differently affect the translation. While bS1-His6 (with a His tag at the C terminal) greatly inhibited translation of rpsA, bS1-dD6 (Domain 6 removed) promoted it. However, both bS1-His6 and bS1-dD6 promoted the translation of rpsO. Our results suggest that bS1 can interact with the 5’ UTR and affect the translation efficiency of mRNA, but the molecular mechanism remains elusive.