利用單分子螢光共振能量轉移技術探討核醣體在rpsO基因轉錄本上對轉譯起始的影響

Observing Translation Initiation of the Ribosome on rpsO Transcript by smFRET

張舒雅(Shu-Ya Chang)

指導教授 ： 温進德

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.02578

摘要

許多mRNA在一般情況下會形成二級結構，然而，當轉譯作用進行時，其二級結構必須被解開使得密碼子能夠被呈現出來。先前研究指出核醣體自己本身在轉譯作用進行時即具有解旋酶(helicase)的活性，可以解開mRNA所形成的二級結構。大腸桿菌中rpsO基因轉錄本的5’未轉譯區(5’UTR)能夠藉由形成假結或雙髮夾結構來調控其本身的轉譯作用，但因為雙髮夾結構的SD(Shine-Dalgarno)序列無法被呈現，所以核醣體只能與假結結構結合並開始轉譯作用的進行。除此之外，在假結結構中只有SD序列是暴露出來的，而起始密碼子下游卻緊鄰二級結構，所以核醣體必須解開下游的二級結構才能完成轉譯起始作用。然而，目前對於假結結構在起始作用中哪個階段被核醣體解開的仍然不清楚。
本篇研究透過單分子螢光共振能量轉移技術來探討當核醣體小次單元及起始tRNA存在時，rpsO基因轉錄本之5’未轉錄區的結構會有何變化。研究結果發現，當核醣體小次單元存在並與mRNA結合時，mRNA形成假結結構的比例會增加，與先前研究核醣體小次單元只能和假結結構結合的結果相符。不只如此，當30S結合到假結結構的SD序列上時，核醣體小次單元能夠藉由解開部分mRNA的二級結構來找到起始密碼子AUG。而當同時有30S及起始tRNA出現時，起始tRNA能夠幫助核醣體小次單元將下游的二級結構全部解開，使得前轉譯起始複合體可以完成。以上研究結果顯示，在轉譯起始階段，核醣體小次單元自己本身即可表現解旋酶活性，將具有二級結構的mRNA解開，讓轉譯作用可以順利進行。

關鍵字

核醣體 ； 轉譯起始作用 ； 單分子 ； 螢光共振能量轉移技術 ； rpsO基因

並列摘要

Many mRNAs fold into secondary structures; however, their codons must be in single-stranded form to be translated. Previous research has revealed that the ribosome itself has helicase activity during the translation process. The rpsO gene transcript of Escherichia coli regulates its own translation through the 5’ untranslated region (5’ UTR), which can fold into a pseudoknot or a double-hairpin conformation. The two structures can be interchanged spontaneously, but the ribosome can only bind to the pseudoknot to initiate translation. On the pseudoknot, only the Shine-Dalgarno (SD) sequence but not the AUG start codon is fully exposed, so the ribosome has to unwind part of the secondary structure to complete the initiation. However, it remains unclear in which
stage the conformation is opened by the ribosome.
In this study, we characterize the conformational change of the rpsO 5’ UTR in the presence of the 30S ribosomal subunit and initiator tRNA (charged formyl-methionyl tRNA) by using single-molecule fluorescence resonance energy transfer (smFRET). Our results show that the population of the pseudoknot form is increased when 30S binds to the RNA. 30S would begin to search for the AUG start codon after binding to the SD sequence of the pseudoknot by partially unwinding the local structures. In the presence of the initiator tRNA, 30S may completely unwind a stem of the pseudoknot and form the pre-initiation complex. These results demonstrate that the 30S ribosomal subunit alone can perform its helicase activity during the initiation stage.

並列關鍵字

ribosome ； translation initiation ； single-molecule ； FRET ； rpsO gene

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