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標題: | 酥胺酸衰減子對T3及SP6RNA聚合酵素轉錄終止效率影響之研究 The Effects of Transcription Termination by T3 and SP6 RNA Polymerases with the Threonine Attenuator |
作者: | Sheue-Hwey Lay 賴雪蕙 |
出版年 : | 1996 |
學位: | 碩士 |
摘要: | 大腸桿菌酥胺酸操縱子(threonine operon)由數個結構基因組成,負責產生由天門冬胺酸(aspartic acid)合成酥胺酸(threonine)的有關酵素,這些基因的表現受到衰減機制(attenuation)的控制。酥胺酸衰減子(thr attenuator)位於酥胺酸操縱子上游調控區域,具有典型rho-independent終止子構造:富含G-C鹼基配對的逆對稱序列,及緊接其後一串由A-T鹼基對構成的區域。轉錄後,酥胺酸衰減子會形成3'端帶有9個U的RNA髮夾形構造(hairpin structure)。 本實驗利用酥胺酸衰減子及其變異株,在構築到T3?動子(promoter)或SP6?動子後,將質體線性化,在α-32P-CTP標定下分別進行離體轉錄,並以含urea之6% polyacrylamide gel分析產物,再進行放射性物質定量並計算轉錄終止效率,藉以瞭解酥胺酸衰減子序列或RNA穩定度對轉錄終止效率之影響。 實驗結果顯示,酥胺酸衰減子及其變異株對T3 RNA聚合酵素之轉錄終止效率較SP6 RNA聚合酵素高,並且RNA髮夾形構造的穩定性及其核?酸序列均會影響T3及SP6 RNA聚合酵素的轉錄終止效率。 此外,本文亦探討酥胺酸衰減子3'端A-T鹼基對區域的系列刪減變異株對T3 RNA聚合酵素轉錄終止效率之影響,結果顯示:酥胺酸衰減子範本上至少需有4個A,對T3 RNA聚合酵素才有些許終止效果(22.8%),而範本上有8個A存在時,則終止效率和野生型衰減子相同(83.3%)。相較於大腸桿菌RNA聚合酵素,噬菌體的RNA聚合酵素需要範本上較多連續A-T鹼基對序列,方可達到最大終止效率。 The threonine operon of Escherichia coli is composed of three structural genes enconding four of the five enzymatic activities required for the synthesis of threonine from aspartic acid. The regulation of the threonine operon appears to occur at the level of transcription termination via an attenuation mechanism. The thr operon regulatory region is structurally similar to a typical factor-independent terminator. It consists of a G+C-rich region of dyad symmetry immediately followed by an A+T-rich region of nine base pairs. When the thr operon regulatory region is transcribed into RNA, the RNA transcript can form a region of secondary structure containing a G+C-rich stem and loop followed by a run of nine uridine residues at the 3' end of the transcript. In this thesis, fifteen thr attenuator variants were analyzed to find the factors which affect the transcription termination of T3 and SP6 RNA polymerases. The results indicate that not only the hairpin structure itself but also its sequence influences termination. In addition, a series of seven deletion variants that successively shorten the deoxyadenosine tract in the attenuator template were analyzed on T3 RNA polymerase. Results from these experiments indicate that complete readthrough occurs when there is one or two deoxyadenosine residues. With 4 template deoxyadenosine residues there is 27% termination increasing to 84% with 8 deoxyadenosines, the value produced by the wild-type attenuator. Compared with E. coli RNA polymerase, bacteriophage RNA polymerases require longer deoxyadenosine tracts than does the bacterial enzyme to terminate with maximum efficiency. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76219 |
全文授權: | 未授權 |
顯示於系所單位: | 植物科學研究所 |
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