臺灣博碩士論文加值系統

在生物體中有許多方式可調控轉譯反應，進而影響蛋白質的產生與否，-1核醣體框架轉移(-1 programmed ribosomal frameshifting, -1 PRF)則是其中一種。-1核醣體框架轉移在許多病毒中可發現，病毒藉由此種機制來調控其所需蛋白質間之比例。要發生-1核醣體框架轉移必須有兩個因素，一為滑動區域(slippery site)，slippery site為一組由7個核&;#33527;酸所組成的特定序列，位於可產生-1核醣體框架轉移RNA之5’端。第二個因素為距滑動區域5-7個核&;#33527;酸下游處的特殊RNA二級結構，稱為stimulator RNA。能作為stimulator RNA的二級結構可能有許多種，如HIV-1的hairpin，BWYV的H-type pseudoknot或是SARS-CoV的three-stem pseudoknot等，而其中最為常見的為pseudoknot類型。
2008年Joy Xin Wang 等人的研究中發現有一特定序列RNA(68 metH RNA)可結合S-adenosylhomocysteine (SAH) 這個代謝產物以做為一個riboswitch，可用來調控S-adenosyl-L-methionine (SAM)循環再生系統。而68 metH RNA二級結構預測為一RNA pseudoknot。之前本實驗室已証實68 metH RNA在加入SAH後的確可引起-1核醣體框架轉移。而我則利用定點突變方式探討68 metH RNA上特定鹼基配對改變對-1核醣體框架轉移效率的影響。實驗數據指出當破壞stem區域鹼基配對導致無法形成stem結構時，即使在最高SAH濃度下也幾乎無法偵測到-1核醣體框架轉移之訊號，所以stem區域對引發-1核醣體框架轉移是必須的。並且若是增加stem 2的穩定性則可使SAH引起的-1框架轉移效率增加，可作為調控68 metH RNA藉由結合SAH引發-1核醣體框架轉移之處。
由於將68 metH RNA進行序列比對後發現有幾個鹼基在不同菌種中具有高度保留性，利用定點突變置換序列後就無法引起-1核醣體框架轉移反應，表示具高度保留性之鹼基扮演很重要的地位。

There are many ways to regulate translation, and -1 programmed ribosomal frameshifting (-1 PRF) is one of them. -1 PRF is found in many type of viruses which can regulate the ratio of essential proteins by -1 PRF. Efficient -1 PRF requires two RNA elements. The first one is a hepta-nucleotide slippery sequence, located in the 5’ end named “slippery site”. The second element is a stimulator RNA structure, located 5-7 nucleotides downstream of the slippery site. There are many kinds of structures that can be a stimulator RNA, such as a simple hairpin in HIV-1, the H-type pseudoknot in BWYV or the three-stem pseudoknot in SARS-CoV. The most common of them is a hairpin-type pseudoknot.
A specific RNA motif named 68 metH RNA was found in 2008. This RNA motif, as a riboswitch, can regulate S-adenosyl-L-methionine (SAM) recycling by binding S-adenosylhomocysteine (SAH). In addition, secondary structure prediction suggested that 68 metH RNA is a pseudoknot. Previously, we have confirmed the 68 metH RNA can induce -1 PRF in response to SAH concentration. I demonstrate here the influence of the base pairs in the 68 metH RNA on SAH-dependent -1 PRF by mutagenesis. When the stem regions was disrupted, no -1 PRF signal could be detected even in the presence of high SAH concentration. Therefore, the 68 metH RNA requires all the stem regions to induce SAH-dependent -1 PRF. Furthermore, I also demonstrate that increasing the stability of stem 2 can improve the SAH-dependent -1 PRF efficiency.
Sequence alignment of SAH riboswitch suggests that several nucleotides are highly conserved. The 68 metH RNA could not induce -1 PRF when these highly conserved nucleotides were mutated. It thus implies that these nucleotides play important roles in SAH-dependent -1 PRF of 68 metH RNA.

目錄......................................................I
圖表目錄................................................III
中文摘要.................................................IV
Abstract..................................................V
第一章 背景介紹..........................................1
1.1 轉譯作用讀取框架位移..................................1
1.2 計劃性-1核醣體框架轉移 (-1 programmed ribosomal frameshifting，-1 PRF)....................................2
1.2.1 -1 PRF在病毒蛋白轉譯作用中扮演之角色................2
1.2.2 -1 PRF產生訊號......................................2
1.2.3 導致發生-1 PRF之stimulator RNA三度空間結構特徵......4
1.2.4 -1 PRF作用機制......................................7
1.3 Riboswitch............................................9
1.3.1 Riboswitch作用機制..................................9
1.3.2 Riboswitch結構.....................................11
1.4 研究動機及目標 .......................................12
第二章 材料與方法 .......................................13
2.1 質體構築.............................................13
2.1.1 聚合酶連鎖反應 (polymerase chain reaction, PCR)....13
2.1.2 勝任細胞(competent cell)製作.......................13
2.1.3 轉型(transformation)...............................14
2.1.4 質體DNA萃取........................................14
2.1.5 構築定點突變質體DNA................................15
2.2 -1 框架轉移效率分析 (-1 frameshift efficiency assay).15
2.2.1 SDS 電泳分析 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE)...........................15
2.2.2試管內轉錄作用製備RNA (in vitro transcription)......16
2.2.3 -1 框架轉移效率分析................................16
第三章 實驗結果.........................................18
3.1 pseudoknot中各stem對68 metH RNA引起之-1 PRF的影響....18
3.1.1 實驗所用SAH motif RNA之二級結構預測................18
3.1.2 破壞stem區域鹼基配對可大幅降低由SAH引起之-1 PRF效率，若復原鹼基配對則可使-1 PRF再次發生.......................18
3.1.3 若將stem區更大範圍鹼基對破壞配對後再加以復原，除P2突變株外皆無法有SAH所引起之-1 PRF反應產生 19
3.2 針對兩次突變不同點進行定點突變並分析-1 PRF效率 19
3.2.1 針對P1部份於兩次stem mutation差異處進行定點突變並分析-1 PRF效率 19
3.2.2 針對P4部份於兩次stem mutation差異處進行定點突變並分析-1 PRF效率 21
3.3 改變P2區域穩定性對-1 PRF效率之影響 22
3.3.1 增加P2 stem後端區域穩定性可提昇-1 PRF效率 22
3.3.2 P2UACG及P4CG突變株皆可提昇-1 PRF效率，卻無法同時加成 22
3.4 slippery site及spacer對 -1 PRF效率的影響 23
3.4.1 不同slippery site序列可提昇 -1 PRF效率 23
3.4.2 不同sapcer長度之pseudoknot無法得到較高的-1 PRF效率 24
3.5. 置換L1核苷酸會導致68 metH RNA無法藉由結合SAH引發-1 PRF反應 24
第四章 討論 26
4.1 68 metH RNA晶體結構與SAM-III riboswitch晶體結構之關聯 26
4.2 stem區域如何影響-1 PRF效率 27
第五章 實驗圖表 29
第六章 附錄 41
附錄1 實驗中所使用引子 41
附錄2 構築突變株所用之引子及突變位 45
附錄3 未進行實驗之突變株示意圖 47
第七章 參考文獻 48

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