臺灣博碩士論文加值系統

在大腸桿菌中有許多非轉譯小型核醣核酸(sRNAs)。這些非轉譯小型核醣核酸可在不同生長環境下表現，並且經由多種機制調控細菌內基因的表現使細菌能迅速適應環境改變。非轉譯小型核醣核酸主要藉由鹼基配對與目標mRNA交互作用，可分為完全配對(cis-encoded)或是部分配對(trans-encoded)。雖然sRNA也可調控基因轉錄，大部分的sRNA作用在轉錄後基因調控(post-transcriptional level)，而促進或是抑制mRNA的降解。這裡我們發現DsrA此sRNA可能具有新的功能可調控外來質體DNA的複製。根據過去研究指出，DsrA具有兩段大約15-20個鹼基的序列可活化或是抑制多個基因。我們利用生物資訊去尋找這兩個序列是否有其他可能的目標並將結果進行分析，發現ColE1 type質體所表現的小分子RNA(antisense RNA) RNAI與DsrA有可能的部分鹼基配對。為了更進一步證實預測結果，我們將表現RNAI但不影響質體DNA複製的pCML108質體送入大腸桿菌中，看有無DsrA表現時對RNAI表現量的影響。我們發現在缺乏DsrA的情況下，RNAI的表現量明顯下降，是因為DsrA可以抑制RNAI降解的速度。接著，我們在過度表現DsrA之下偵測質體DNA的量，發現ColE1 type質體DNA的數目下降。另一方面，我們發現，將質體(pCM128)送入大腸桿菌中會造成DsrA的表現穩定，而送入表現RNAI的質體(pCML108)能使得DsrA穩定性更加上升。我們認為大腸桿菌表現的DsrA與質體表現的RNAI之間會相互調控。兩者之間相互作用的分子機制以及協助sRNA與目標基因配對的RNA chaperone Hfq蛋白在之中所扮演的角色將會更進一步探討。根據實驗的結果，我們發現一個全新的sRNA調控機制，是大腸桿菌內的sRNA與外來質體DNA的sRNA之間的交互作用。

Several small non-coding RNAs (sRNAs) have been identified in E. coli that act by various mechanisms and are involved in a wide range of physiological responses. The majority of sRNAs characterized to date interact with mRNA targets by base pairing, having either extensive (cis-encoded) or limited (trans-encoded) complementarities with their targets. Although sRNAs are involved in transcriptional regulation, they primarily act on their targets at the post-transcriptional level, which may result in destabilization or stabilization of mRNAs. Here we discovered that the sRNA DsrA may play a role in response to incoming plasmid and inhibit DNA plasmid replication in bacteria. It is known that two different regions in the DsrA sequence ranging from 15 to 20 nucleotides can target multiple transcripts with different modes of regulations. Searching for sequence complementarities of these two DsrA regions with known sRNAs in E. coli, we found that the antisense RNA RNAI of ColE1-type plasmids has a putative binding site for DsrA. By introducing a non-ColE1 type plasmid expressing RNAI named pCML108 into E. coli MG1655 wild-type and 岛dsrA strains, we found a decrease in RNAI expression as well as in RNAI stability in the 岛dsrA strain. Overexpression of DsrA also decreases the ColE1 plasmid copy number. We also found that introducing pCM128 plasmid (without RNAI expression) into E.coli can prolong the stability of DsrA, and introducing pCML108 (with RNAI expression) prolongs DsrA stability even more. Whether the RNA chaperone Hfq plays a role in this DsrA-RNAI regulation will be further addressed. The regulatory mechanism between these two sRNAs will be further examined by mutating the putative binding sites. In summary, this finding reveals a novel sRNA regulatory role.

Acknowledgment (Chinese)…………………………………………………………..i
Table of contents……………………………………………………………………..ii
List of tables and figures…………………………………………………………….iii
Abstract (Chinese)……………………………………………………………………1
Abstract (English)…………………………………………………………………….2
Introduction …………………………………………………………………………3
Materials and Methods………………………………………………………………10
Results………………………………………………………………………………15
Discussion……………………………………………………………………………20
References……………………………………………………………………………26
Tables and Figures……………………………………………………………………35
Appendix…………………………………………………………………………49

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