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標題: | 以光鉗技術探討核醣體在rpsO基因轉錄本上對轉譯起始的影響 Observing Translation Initiation of Ribosomes on the rpsO Transcript using Optical Tweezers |
作者: | Cheng-Wen Hsi 奚正文 |
指導教授: | 溫進德(Jin-Der Wen) |
關鍵字: | 核醣體,轉譯起始作用,單分子技術,雷射光鉗,rpsO 基因,偽結結構,雙髮夾結構, ribosome,translation initiation,single-molecule technique,optical tweezers,rpsO gene,pseudoknot,double-hairpin, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 在原核生物中,核醣體在轉譯起始的過程中必須先辨識mRNA 5’端未轉譯區(5’UTR)上的Shine-Dalgarno sequence (SD 序列),進而使起始tRNA 配對到起始密碼子,轉譯方能開始。許多單股mRNA 在一般情況下會形成二級結構,在轉譯起始的過程中,這些二級結構必須被解開使密碼子能夠被呈現出來。大腸桿菌中rpsO 基因轉錄本5’UTR(rpsO 5’UTR)能夠藉由形成雙髮夾結構或偽結結構來調控自身序列的轉譯作用,但因為SD 序列被隱藏在雙髮夾結構中,所以核醣體只能結合在偽結結構開始轉譯作用。然而,在偽結結構只有SD 序列是暴露出來的,起始密碼子下游緊鄰另一個二級結構,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。
本篇研究經由單分子光鉗技術來探討當核醣體小次單元30S 及起始tRNA 存在時,對rpsO 5’UTR 的結構會有何影響。透過研究結果發現,和30S 反應過後的rpsO 5’UTR 和未加入30S 的組別,在光鉗實驗中看不出明顯差異。當同時有加入30S 及起始tRNA 的情況時,rpsO 5’UTR 下游的二級結構會被全部解開。刪減rpsO 5’UTR 兩個髮夾間的核苷酸數量,在光鉗實驗發現核醣體30S結合至RNA 的分子比例降低,在細胞體外轉譯實驗中得知刪減核苷酸數量的組別在轉譯效率上明顯下降。綜合以上結果,在轉譯起始階段30S 和起始tRNA 能夠解開rpsO 5’UTR 下游結構,且雙髮夾間的核苷酸扮演了30S 和起始tRNA 結合RNA 重要的角色。 In prokaryotes, ribosomes have to recognize the Shine-Dalgarno sequence (SDsequence) on mRNA 5’UTR, and then the initiator tRNA can pair to the start codon to initiate translation. Many mRNAs fold into secondary structures in normal situation.During the translation initiation process, these secondary structures have to be unfolded to revel the start codon. The rpsO gene transcript 5’ untranslated region (5’UTR) of Escherichia coli regulates its own translation through folding into a doublehairpin or a pseudoknot conformation. However, the SD sequence is concealed in the double-hairpin structure. Ribosomes can only bind to pseudoknot conformation to initiate translation. The SD sequence is fully exposed only on the pseudoknot. The start codon is adjacent to the downstream secondary structure, so the ribosome has to unwind the secondary structure to complete the initiation process. In this research, we investigate the conformational change of the rpsO 5’ UTR in the presence of the ribosomal subunit 30S and initiator tRNA by using optical tweezers. Our results showed that there was no significant difference for the rpsO 5’UTR treated with or without 30S. In the presence of the 30S and initiator tRNA, rpsO 5’ UTR downstream secondary structure could be completely unfolded. The optical tweezers’ data showed that nucleotides deletion between the two hairpins decreased the 30S binding probability to RNA molecules, and also decreased the in vitro translation efficiency. These results demonstrate that the 30S and initiator tRNA can unfold the rpsO 5’ UTR downstream secondary structure, and the length of nucleotides between the two hairpins plays an important role in binding of 30S and tRNA to rpsO 5’ UTR. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68822 |
DOI: | 10.6342/NTU201703738 |
全文授權: | 有償授權 |
顯示於系所單位: | 分子與細胞生物學研究所 |
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