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  本文關(guān)鍵詞： 核酶 RNase P M1GS 人巨細胞病毒HCMV DNA聚合酶　出處：《暨南大學》2005年碩士論文　論文類型：學位論文

【摘要】：大腸桿菌來源的RNase P是特異性剪切前體tRNA(ptRNA)的5′端,使之成熟的天然核酶,該酶的催化核心為一個377nt的RNA亞單位(M1RNA),具有獨立剪切RNA的核酶活性。RNase P是結(jié)構(gòu)識別酶,RNase P的模式底物至少包括兩個要件:1 游離NCCA-3′術(shù)端;2 特異互補的雙鏈RNA區(qū)域。人為設計的引導序列GS(guide sequence)與mRNA形成RNase P能夠識別的底物形式。人工核酶M1GS就是由大腸桿菌來源的RNase P的催化中心M1RNA與引導序列GS共價連接而成的新型核酶,在引導序列GS引導下可實現(xiàn)對模式底物的特異切割。 本課題以人巨細胞病毒(HCMV)DNA聚合酶基因UL54為靶基因,針對該基因構(gòu)建了人工核酶M1GS-T7,并從M1GS-T7的二級結(jié)構(gòu)和體外切割活性兩方面進行研究,從而探索M1GS-T7結(jié)構(gòu)和活性的對應關(guān)系,為進一步研究M1GS的功能和結(jié)構(gòu)奠定理論基礎(chǔ)。 構(gòu)建了兩種不同基因組成的人工核酶M1GS-T7,M1GS-T7bridge,以研究連接序列(bridge)對M1GS活性的影響,兩種轉(zhuǎn)錄模板DNA分別包括有T7啟動子、M1RNA基因、bridge序列以及GS序列四個組成部分的M1GS-T7bridge和不包括連接序列的M1GS-T7。RNA二級結(jié)構(gòu)模擬顯示:在無連接序列的M1GS-T7中,GS參與了M1RNA二級結(jié)構(gòu)的形成,從而破壞了M1RNA的活性構(gòu)象;而加入了連接序列的M1GS-T7bridge,維持了M1RNA的獨立折疊。體外切割結(jié)果顯示:有連接序列的M1GS-T7bridge仍然有體外切割活性,無連接序列的M1GS-T7失去體外切割活性。 模擬了M1GS-T7bridge在不同溫度下的二級結(jié)構(gòu),模擬結(jié)果顯示:溫度降低到20℃時,M1RNA的各個螺旋區(qū)域進行重排;而當溫度升高到55℃時,M1RNA的結(jié)構(gòu)中,對M1RNA維持活性必需的,且與底物CCA末端直接作用的重要螺旋發(fā)生了變化。體外切割實驗結(jié)果顯示,20℃ M1GS-T7bridge無活性了,而55℃相比37℃下的活性高了。說明在M1RNA中,催化活性部位相對非活性部位有更強的敏感性,活性部位的結(jié)構(gòu)變化對應著核酶活性的高低變化,非活性部位的變化對應著核酶活性的有無。 為一步證實55℃下,M1GS-T7bridge活性的提高是由于二級結(jié)構(gòu)的改變,根據(jù)RNA二級結(jié)構(gòu)模擬,引入了突變G~(250)G~(251)G~(260)G~(275)到T~(250)T~(251)A~(260)C~(275),使突變后的mM1GS-T7bridge的37℃下的二級結(jié)構(gòu)與野生型M1GS-T7bridge的55℃的結(jié)構(gòu)一致;體外切割檢測兩者的活性,結(jié)果顯示,突變型核酶比野生型核酶活性略高,證明核酶的二級結(jié)構(gòu)與其活性之間有嚴
[Abstract]:E.coli-derived RNase P is a specific shear precursor tRNA (ptRNA) of the 5 'end of the mature natural ribozyme, the RNA subunit of the enzyme catalytic core as a 377nt (M1RNA),.RNase P ribozyme with independent RNA shear structure is the recognition model of enzyme, substrate RNase P include at least two elements: 1 free NCCA-3' operation end; 2 specific complementary double stranded RNA region. The design of artificial guide sequence GS (guide sequence) RNase P can recognize the substrate form and mRNA. M1GS is composed of artificial ribozyme catalytic center from Escherichia coli RNase P M1RNA and GS covalently linked guide sequence a new ribozyme, the boot sequence under the guidance of GS can realize the specific mode of substrate cutting.
This subject to human cytomegalovirus (HCMV) DNA polymerase gene UL54 as the target gene, the gene construct artificial ribozyme M1GS-T7, and from the two levels of structure and in vitro M1GS-T7 cleavage activity were studied in two aspects, so as to explore the relationship between M1GS-T7 activity and structure, to lay the theoretical foundation for the further study on the structure and function of M1GS.
Constructed two different genes consisting of artificial ribozyme M1GS-T7, M1GS-T7bridge, to study the connection sequence (bridge) effect on the activity of M1GS, two DNA respectively, including transcription template T7 promoter, M1RNA gene, bridge sequence and GS sequence of the four components of M1GS-T7bridge and does not include analog connection sequence M1GS-T7.RNA two level structure display: in the connectionless M1GS-T7 sequence, GS is involved in the formation of M1RNA two level structure, thus destroying the active conformation of M1RNA; and joined the connection sequence of M1GS-T7bridge, to maintain the M1RNA independent folding. In vitro cleavage results showed that: M1GS-T7bridge still have a connected sequence of cleavage activity in vitro, no connection sequence M1GS-T7 lost the cleavage activity in vitro.
Simulation of the two level structure of M1GS-T7bridge at different temperatures. The simulation results show that the temperature is reduced to 20 DEG C, the rearrangement of each helix region of M1RNA; and when the temperature rises to 55 degrees, the structure of M1RNA, M1RNA on the activity of the necessary changes, and an important substrate CCA and spiral end directly in vitro cleavage effect. The experimental results show that M1GS-T7bridge 20 degrees of activity, and 55 degrees compared to 37 DEG C high activity. In the M1RNA, the catalytic active site relative to non active site has higher sensitivity, structural changes in the active site of the corresponding changes of enzymatic activity, the active site of the corresponding non change a ribozyme is there.
For a further 55 DEG C, the activity of M1GS-T7bridge was improved by two grade structure change, based on the simulation of RNA two level structure, introduces the mutation of G~ (250) G~ (251) G~ (260) G~ (275) T~ (250) T~ (251) A~ (260) C~ (275). The structure of two level structure after the mutation of mM1GS-T7bridge at 37 DEG C and wild type M1GS-T7bridge and 55 DEG C; the detection of in vitro cleavage activity, results showed that the mutant ribozyme is slightly higher than the wild-type ribozyme, between the two level structure of the ribozyme and its activity is strictly proved

【學位授予單位】：暨南大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：Q789

【引證文獻】

相關(guān)碩士學位論文 前1條

1 李璽洋;草魚形態(tài)性狀對體質(zhì)量影響效果分析及生長相關(guān)SNP位點篩選[D];華中農(nóng)業(yè)大學;2012年

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本文編號：1539781