M1GS功能结构位点的初步研究

发布时间：2018-02-26 21:41

本文关键词： 核酶 RNase P M1GS 人巨细胞病毒HCMV DNA聚合酶　出处：《暨南大学》2005年硕士论文　论文类型：学位论文

【摘要】：大肠杆菌来源的RNase P是特异性剪切前体tRNA(ptRNA)的5′端,使之成熟的天然核酶,该酶的催化核心为一个377nt的RNA亚单位(M1RNA),具有独立剪切RNA的核酶活性。RNase P是结构识别酶,RNase P的模式底物至少包括两个要件:1 游离NCCA-3′术端;2 特异互补的双链RNA区域。人为设计的引导序列GS(guide sequence)与mRNA形成RNase P能够识别的底物形式。人工核酶M1GS就是由大肠杆菌来源的RNase P的催化中心M1RNA与引导序列GS共价连接而成的新型核酶,在引导序列GS引导下可实现对模式底物的特异切割。本课题以人巨细胞病毒(HCMV)DNA聚合酶基因UL54为靶基因,针对该基因构建了人工核酶M1GS-T7,并从M1GS-T7的二级结构和体外切割活性两方面进行研究,从而探索M1GS-T7结构和活性的对应关系,为进一步研究M1GS的功能和结构奠定理论基础。构建了两种不同基因组成的人工核酶M1GS-T7,M1GS-T7bridge,以研究连接序列(bridge)对M1GS活性的影响,两种转录模板DNA分别包括有T7启动子、M1RNA基因、bridge序列以及GS序列四个组成部分的M1GS-T7bridge和不包括连接序列的M1GS-T7。RNA二级结构模拟显示:在无连接序列的M1GS-T7中,GS参与了M1RNA二级结构的形成,从而破坏了M1RNA的活性构象;而加入了连接序列的M1GS-T7bridge,维持了M1RNA的独立折叠。体外切割结果显示:有连接序列的M1GS-T7bridge仍然有体外切割活性,无连接序列的M1GS-T7失去体外切割活性。模拟了M1GS-T7bridge在不同温度下的二级结构,模拟结果显示:温度降低到20℃时,M1RNA的各个螺旋区域进行重排;而当温度升高到55℃时,M1RNA的结构中,对M1RNA维持活性必需的,且与底物CCA末端直接作用的重要螺旋发生了变化。体外切割实验结果显示,20℃ M1GS-T7bridge无活性了,而55℃相比37℃下的活性高了。说明在M1RNA中,催化活性部位相对非活性部位有更强的敏感性,活性部位的结构变化对应着核酶活性的高低变化,非活性部位的变化对应着核酶活性的有无。为一步证实55℃下,M1GS-T7bridge活性的提高是由于二级结构的改变,根据RNA二级结构模拟,引入了突变G~(250)G~(251)G~(260)G~(275)到T~(250)T~(251)A~(260)C~(275),使突变后的mM1GS-T7bridge的37℃下的二级结构与野生型M1GS-T7bridge的55℃的结构一致;体外切割检测两者的活性,结果显示,突变型核酶比野生型核酶活性略高,证明核酶的二级结构与其活性之间有严
[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

【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q789

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