The Preliminary Study Of M1GS' Functional Domain

RNase P from E. Coli is a ribonucleoprotein complex responsible for the 5' maturation of ptRNAs. The core subunit,M1RNA, is able to cleave the interest RNA on itself. Studies on RNase P substrate recognition reveal that M1RNA recognize the structure rather than the primary nucleotide sequence of the substrates, and can cleave a model substrate that contains two important parts: NCCA-3' terminal and RNA helix area. Thus, M1RNA can cleave a mRNA sequence if the mRNA substrate forms a hybrid complex with its complementary sequence (external guide sequence, EGS) to resemble a pre-tRNA molecule. The EGS can be covalently linked to M1RNA, the catalytic RNA subunit of RNase P, and form a new kind of ribozyme, M1GS.M1GS can cleave any RNA segments in vitro when the interest RNA is in a complex with its guide sequence(GS).We construct M1GS-T7 , which target the mRNA segment of HCMV UL54 gene, and investigate their structure and activity in vitro. Consequently , we could learn the consistent relation of structure and activity. Our studies make the theory base for the research of M1GS' structure and activity.We construct two ribozyme:M1GS-T7 and M1GS-T7bridge. The transcription template of M1GS-T7bridge include:T7 promoter, M1RNA gene, bridge and GS sequence,while M1GS-T7 doesn't contain bridge. The RNA secondary structure simulation demonstrate that guide sequence form a hybrid complex with M1RNA and M1RNA could not form the right conformation , so M1GS lose the activity;while the existence of bridge keep M1RNA fold naturally. In vitro cleavage result indicates that M1GS-T7 bridge can cleave the substrate efficiently; However, M1GS-T7 without bridge don't show activity of cleavage. It confirms that the bridge contribute to the completeness of confirmation of M1RNA, and play an important role in keeping the M1GS-T7's activity of in vitro cleavage.I simulate the secondary structure of M1GS-T7bridge under different temperature.The result show: when it degrades to 20℃,the secondary structure of M1GS-T7bridge changes so greately that important double helix can't form rightly and the activity lose;however, when it rise to 55℃,only the double helix which interact with CCA of substrate change ,We explore the connection betweentemperature and activity, the in vitro cleavage result reveal that M1GS-T7bridge is inactive in 20°C, while the activity of M1GS-T7bridge rise a little in 55°C.The result demonstrate that the catalytic center is more gentle than the other structure, the former affect the active degree of ribozyme, while the latter will lead to inactive.To elucide the cau of M1GS-T7bridge' s rised activity, I introduct a mutation of G250G251G260G275 to T250T25lA260C275, then the structure of mutant M1GS-T7bridge in 37 °C agree with wild type M1GS-T7bridge in 55°C.The result of in vitro cleavage tells us the mutant type is more active than MlGS~T7bridge. The experiment demonstrate that the structure of ribozyme is in correspondence with its activity strictly.Our study succeedes to pridicate the structure of active and the inactive MIRNA of M1GS-T7 with bioinformation, the methods provide important preference for the design of the efficient M1GS.