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標題: | 大腸桿菌中ColE1質體上調控型核醣核酸RNAI的非典型功能 Noncanonical function of ColE1-type plasmid regulator RNAI in E.coli |
作者: | Wei-Syuan Wang 王唯萱 |
指導教授: | 林淑端(Sue Lin-Chao) |
關鍵字: | 調控型核醣核酸,Hfq蛋白,RNAI,質體-細菌的影響,大腸桿菌, sRNA regulation,Hfq,RNAI,plasmid-host interaction,E. coli, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 現今科學研究中,質體常用來做為基因表現、基因重組的工具。然而,質體的存在會造成細菌的代謝壓力,因此,質體的數量必須要被嚴密的調控。ColE1 型質體的複製是從引子核醣核酸 RNAII 的轉錄開始,用以做為去氧核醣核酸複製的引子。另一方面,反股核醣核酸 RNAI 會與 RNAII 進行鹼基配對以防止 RNAII 與質體雜合而抑制質體複製。雖然先前研究已發現質體會影響細菌的代謝以及改變其
基因表現,然而,其詳盡的調控機制目前仍不清楚。調控型微小核醣核酸可分為三個功能區,分別為種子區域(seed region)、伴護蛋白 Hfq 結合區、及終結子(ρ-independent terminator)。RNAI 也帶有相同終結子,我們首先想利用 RNA-seq 技術了解是否質體上的反股核醣核酸 RNAI 能夠改變宿主的基因表現。實驗發現宿主的 4497 個基因中有 177 個基因會因為 RNAI 的存在而有兩倍以上的表現量差異。利用 EMSA 技術發現 RNAI 與 Hfq 有高度結合力。利用突變的 Hfq 蛋白進一步分析,我們發現 Hfq 近端面突變會影響RNAI 與 Hfq的結合。另外,細菌本身的調控型核醣核酸會與 RNAI 競爭 Hfq 蛋白的結合。最後,利用定量聚合脢連鎖反應(qPCR)技術,我們進一步確認 RNAI 對於基因表現的調控需要 Hfq 的參與。綜合上述實驗,我們認為 RNAI 可能和微小型核醣核酸以相同方式作用,且其功能需要 Hfq 蛋白的參與。當 RNAI 存在時,RNAI 可能會與調控型核醣核酸競爭 Hfq 間接改變原有的基因表現調控或是在 Hfq 的幫助下能直接調控細菌的基因表現。 Plasmid, a small self-replicated extra-chromsomal DNA element, is widely used in scientific research nowadays. However, introduction of plasmids causes metabolic burden to cells, and therefore, the plasmid copy number needs to be tightly regulated. ColE1-type plasmid replication is initiated by primer RNAII transcription, which serves as primer for DNA polymerase to execute DNA synthesis. On the other hand, the antisense regulator RNAI negatively controls ColE1 plasmid replication by base paring with RNAII, which prevents RNAII-DNA hybrid formation. Although it has been known that upon plasmid-host interaction, cell will have metabolic shift and change gene expressions, however, the underlying mechanism is still unknown. The endogenous sRNAs have 3 functional elements including the seed region, the Hfq binding site, and the ρ-independent terminator. Since RNAI has also ρ-independent terminator, we first used RNA-seq to investigate whether the plasmid-born antisense regulator RNAI also can change gene expression of host. We found that RNAI itself changes host gene expression more than 2 folds for 177 genes over 4497 genes tested. By using in vitro electrophoretic mobility shift assay (EMSA), we found that RNAI binds to Hfq with submicromolar binding affinity. Furthermore, by using Hfq proximal and distal face mutants, we found that only proximal face mutations disrupted RNAI binding. This data suggests that similar to most endogenous sRNAs, RNAI also bind to the proximal face of Hfq. Competition assay shows that RNAI competes with endogenous sRNAs for binding. By using quantitative PCR, we further show that the effects of RNAI on host gene expression depend on the existence of Hfq. In this study, we suggest that Hfq-dependent sRNA-like function could be one of the possible mechanism of how RNAI affects host gene expression. Introduction of RNAI might affect host gene expression either indirectly by competing Hfq binding or by directly targeting to host genes aided by Hfq. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/58685 |
全文授權: | 有償授權 |
顯示於系所單位: | 分子醫學研究所 |
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