Targeted Action Of Human Cytomegalovirus (HCMV) UL54 MRNA Segments In Vitro By The Mutation M1GS Of RNaseP From Escherichia Coli

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.