【作者】 周乐平；

【导师】 李弘剑；

【作者基本信息】 暨南大学 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 大肠杆菌来源RNase P是催化切割ptRNA 5′端成熟的天然核酶，该酶的催化核心是一个377nt的RNA亚单位（M1 RNA），能在具备高盐离子和适当Mg²⁺的体外缓冲环境中对RNA底物进行特异的切割。为了研究M1RNA上位点G²²⁴G²²⁵突变为AA后对核酶作用的影响，我们做了一系列的体外实验来进行研究。 将引导序列（GS）与M1RNA共价连接起来，构成具有特异切割作用的一种核酶M1GS。我们实验室已经成功构建和证明这种核酶对巨细胞病毒（HCMV）UL54基因的mRNA有特异切割的切割作用。UL54基因是负责编码HCMV的DNA聚合酶，这种聚合酶对病毒的复制有重要作用。在本实验中，筛选了两种M1GS核酶，T7-M1GS和C6-M1GS，这两种核酶分别针对UL54基因mRNA的亚克隆片断D和C有特异切割作用。将这两种核酶的M1RNA上224，225位点上的GG突变为AA，并将M1GS克隆到质粒载体pUC18上，获得了两组核酶基因；T7-M1GS，T7-mutM1GS和C6-M1GS，C6-mutM1GS。根据底物片断上的切割位点将UL54基因片段进行亚克隆，构建质粒PU54-A，PU54-B，PU54-C和PU54-D。在我们的实验中，选用质粒PU54-D和PU54-C作为底物模板。 为了研究核酶的作用机制，并且探讨个别碱基对核酶作用的影响，我们比较了突变核酶和野生核酶对他们相应的底物片断的作用。实验结果表明T7-mutM1GS和T7-M1GS这两种核酶对于底物D片断切割效率基本一致，但是突变核酶与底物的结合效率要比野生核酶至少高出2倍。C6-mutM1GS和C6-M1GS对底物C片断的作用也得出类似的结果。 通过分析突变核酶M1RNA的高级结构，发现它的突变位点位于和底物结合的区域，从而加强该核酶与底物的结合，但是这两个突变碱基对核酶的切割没有显著的作用。因此我们认为，这两个位点处在核酶与底物结合的活性中心，并且AA碱基比CC碱基的结合作用要强；核酶的结合作用和切割作用分属两种不同的催化机制，本文进一步证实了有关核酶催化机制的理论结果。更多还原

【Abstract】 RNase P from E.Coli is a ribonucleoprotein complex responsible for the 5’maturation of tRNAs. By linking the ribozyme covalently to a guide sequence(GS) complementary to a targe RNA,the catalytic RNA(M1RNA) can ben converted into a sequence-specific ribozyme,MlGS RNA.We have previously shown that M1GS RNA can efficiently cleave the mRNA sequence encoding DNA polymerase (UL54) of Human cytomegalovirus(HCMV). The UL54 mRNA sequence encoding HCMV DNA polymerase is very important for virus’ duplication.We selected two kinds M1GS RNA,T7-M1GS and C6-M1GS which specific cleave UL54-D mRNA and UL54-C mRNA carried a common mutation at nucleotides 224 and 225 of RNase P catalytic MIRNACG22^225 -> AA).Pucl8,which we chosen as the vector for M1GS gene, was constructed two teams plasmids named T7-MlGS,T7-mutMlGS and C6-MlGS,C6-mutMlGS. pGEMSz, which contains a T7 promoter region, was chosen as the vector for gene UL54. The recombinated plasmid was named PU54. Based on this material, 4 recombinated plasmids PUS4-A, PU54-B, PU54-C and PU54-D were construted, which contained different cloning segments of UL54 gene.In this study we chosen plasmids PU54-D and PU54-C as substrute gene tempers.The mutation ribozyme T7-M1GS and C6-M1GS RNA exhibited as same as cleavage efficiency while 2 times higher binding efficiency than that derived from the wild type ribozyme T7-mutMlGS and C6-mutMlGS RNA.Our results suggest that the mutated A224A225are in close proximity to the substrate binding of the ribozyme .While the function of A224A225 did not significantly affect the activity of the ribozymes to cleave substrate.In this study,experiment using comparing mutation and widetype ribozyme M1GS was carried out to identify the ruction of nucleotides 224 and 225 that enhance ribozyme binding but not cleacage efficient.Identification of the region that interact with mRNA substrate will allow us to study how M1GS RNA recognizes a mRNA substrate and approve the theoretics of ribozyme catalysis.更多还原