臺灣博碩士論文加值系統

轉錄終止是轉錄過程中RNA聚合酶從轉錄複合體脫離的一個重要機制。有許多因子如RNA聚合酶的種類、終止子的結構與受質的濃度會影響轉錄終止。本研究主要以E. coli白胺酸終止子所衍生出來的各種突變體構築為模板，探討T7與葉綠體RNA聚合酶如何在限制核苷酸濃度及終止子的序列改變下影響轉錄終止？白胺酸終止子為典型的rho-independent終止子，具有GC鹼基所組成的莖環結構，及下游連續的T鹼基序列。以連續T鹼基序列的第一個T定為+1，其序號向3′端依序增加以利終止子序列改變位置之描述及討論。T7 RNA聚合酶轉錄結果顯示在限制[rNTP]時，連續T鹼基的長度會影響轉錄終止效率。當終止子的連續T鹼基長度為七個時，在限制[UTP]為5 μM的情況下，轉錄終止效率為最高。而同樣的環境下，連續T鹼基長度為四個T時，該終止子為能夠觀察到最低轉錄終止訊號的結構。由上述連續T鹼基縮短之生體外轉錄終止分析顯示，位在+8與+9鹼基位置上之特定核苷酸會受其核苷酸濃度之限制，而造成轉錄終止之重要改變。因此對+8鹼基位置進行核苷酸種類之更換，並以不同[rNTP]測試+8鹼基位置對轉錄終止效率之影響。結果顯示該位置為A、C、G時，其轉錄終止效率會隨該核苷酸濃度之降低而上升。然而當+8位置為T時，其轉錄終止效率隨[UTP]濃度之減少而降低，推論此現象可能因模板出現連續T時，在高濃度[UTP]之下會造成錯誤聚合移位動作而造成轉錄終止之增加。另一個實驗將T4AT在+8、+9、+10位置上連續的C鹼基，依序分別由A鹼基取代，探討對轉錄終止之影響，結果顯示對轉錄終止的影響，以+8、+9位置的影響較+10位置大。而+9位置的A鹼基在限制[ATP]時，有較高的轉錄終止效率。本研究也利用T3AT在+7與+8位置上分別塞入一個T鹼基，再探討塞入T鹼基後，在限制[UTP]的情況下，對轉錄終止的影響。結果發現+8塞入一T鹼基，可使原本不造成轉錄終止之T3AT產生約46.3%之轉錄終止效率。本研究另外利用葉綠體RNA聚合酶進行轉錄終止之研究，結果顯示與T7 RNA聚合酶在限制[rNTP]的情況下有相類似之結果。唯葉綠體RNA聚合酶在限制[rNTP]的情況下，+10位置較為關鍵。由以上結果推論白胺酸終止子的二級結構與莖環結構下游序列特定位置在限制特定核苷酸濃度反應下皆會影響轉錄終止效率。

Transcription termination involving in the dissociation of the RNA polymerase from the transcription complex is an important process during transcription. There are many factors, such as the type of RNA polymerase, the structure of the terminator and the concentration of substrates, affecting the termination of transcription. In this study, how does the limited concentration of nucleotides and the sequences changed in the terminator affect the transcription termination was examined by T7 and chloroplast RNA polymerases using various mutants created in the E. coli leucine terminator. The leucine terminator is a typical rho-independent terminator containing a structure with a GC-rich stem-loop following by a poly T-tract. The poly T numbering starts as +1 from the first T, then add 1 to every next nucleotide thereafter toward the 3’ end of the terminator. Transcription by T7 RNA polymerase showed that the length of the T-tract affect the termination efficiency under limited concentration of nucleotide [rNTP]. The terminator contains 7 Ts in the T-tract revealed the highest efficiency of termination in the condition where [UTP] was reduced to 5 M, while 4 Ts in the T-tract was the minimum T required for termination under the same condition. An observation from the T-tract-shorten study revealed that the specific nucleotide at the position +8 or +9 was critical when transcription was conducted in vitro at the condition where the same type of [rNTP] was limited. In order to test this observation, mutants with different nucleotides appeared at the position +8 was created. Results of this study confirmed that the increasing of termination efficiency was concomitant with the decreasing concentration of the same type of nucleotide appeared at the position +8. On the contrary, the termination efficiency of terminator T7TT showed proportional to the increased of [UTP]. It can be partly explained by the phenomenon called “molecular slippage” which occurred at the template where a long repeats of the same nucleotide was appeared. This makes the incorporation of the coming nucleotide into the wrong position during high concentration of its substrate is provided in the transcription reaction. A series replacement of “C” by “A” at positions +8, +9 and +10 of T4AT were created to study the positional effect of C on termination. It appeared that position +8 and +9 have been affected more than the position +10. Surprisingly, the mutant containing an “A” at the position +9 resulted in much higher termination efficiency than the T4AT while limiting the [ATP] in the reaction. Another experiment inserting an extra T in the position +7 or +8 of T3AT further demonstrated the importance of +8 position in transcription termination when limiting [UTP]. Moreover, transcription by chloroplast RNA polymerase revealed similar [rNTP] limiting effect to that of T7 RNA polymerase, except the critical position that responded to the [rNTP] is at +10. The results mentioned above suggested that the secondary structure, the sequences at the specific position downstream from stem loop of leucine terminator, and the limited concentration of specific nucleotides in the reaction all affect the efficiency of transcription termination.

目錄 1
中文摘要 3
Abstract 5
壹、前言 7
貳、前人研究 8
一、基因轉錄之分子機制 8
二、RNA聚合酶種類及特性 9
三、轉錄過程之阻礙 11
四、滑動機制的發生 13
五、原核生物的轉錄終止機制 14
六、葉綠體的轉錄終止 15
參、材料與方法 16
一、小量質體DNA的抽取 16
二、大量質體DNA的抽取 17
三、回收純化之DNA片段 18
四、黏接反應 ( ligation reaction ) 19
五、大腸桿菌勝任細胞之製備 ( competent cell preparation ) 19
1﹒適用於熱休克法之製備 ( heat-shock preparation ) 20
2﹒適用於電穿孔法之製備 ( electroporation preparation ) 20
六、轉型作用 ( transformation ) 21
1﹒熱休克法 ( heat-shock method ) 21
2﹒電穿孔法 ( electroporation mehod ) 21
七、聚合酶鏈反應 ( polymerase chain reaction ) 22
八、菠菜葉綠體高鹽萃取液之抽取 22
九、生體外轉錄試驗 24
十、生體外轉錄物之萃取 25
十一、聚丙稀醯胺膠體之製備及電泳分離 25
十二、自動放射顯影 26
十三、轉錄終止訊號之定量計算 26
肆、結果 27
一﹒白胺酸終止子突變體之構築 27
1﹒含不同長度T-tract之構築 27
2﹒T-tract下游+8鹼基位置之單點突變構築 28
3﹒T4AT終止子下游序列GCCCG中C鹼基之單點突變構築 28
4﹒T3AT終止子下游序列特定+7及+8位置崁入T鹼基之構築 29
5﹒pLSTleuTa刪除-80起動子之T-tract之構築 29
二﹒白胺酸終止子生體外轉錄終止分析 30
1﹒含不同長度T-tract生體外轉錄終止分析 30
2﹒T-tract下游序列+8 鹼基位置生體外轉錄終止分析 31
3﹒不同rNTPs梯度濃度對終止子+8鹼基轉錄終止之影響 32
4﹒DNA模板、RNA聚合酶及標定同位素含量之不同對轉錄終止之影響 33
5﹒T4AT終止子下游+8∼+10之CCC鹼基對轉錄終止之影響 33
6﹒T3AT終止子下游+7及+8鹼基位置加入T後對轉錄終止之影響 34
三﹒葉綠體RNA polymerase之生體外轉錄分析 35
1﹒含不同長度T-tarct之生體外轉錄終止分析 35
2﹒T-tract下游序列受rNTPs濃度轉錄終止分析之影響 36
四﹒E coli RNA聚合酶生體外轉錄定性分析 37
五﹒Thermus RNA聚合酶生體外轉錄定性分析 37
伍、討論 38
一﹒白胺酸終止子T-tract長度及下游序列在限制rNTPs濃度下會影響轉錄終止效率 38
二﹒終止子下游+8、+9位置之鹼基在限制rNTPs濃度下影響轉錄終止效率 39
三﹒白胺酸T7TT終止子隨UTP濃度之增加，轉錄終止效率增加可能是因轉錄滑動現象所造成 40
四﹒T3AT終止子下游特定位置在限制rNTPs濃度下影響轉錄終止效率 41
五﹒葉綠體RNA聚合酶轉錄終止受rNTP濃度之影響 42
六﹒E. coli RNA聚合酶與耐熱性RNA聚合酶的生體外轉錄終止分析 43
陸、參考文獻 45
柒、圖表 53
捌、附錄 75

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