Preliminary Study On The Function Of PPR Protein

RNA editing is the process of changing the genetic information encoded by DNA by replacing,inserting or deleting bases at a specific position on the m RNA.RNA editing is widely present in animals and plants,effectively expanding genetic information,which is the reason that organisms can better adapt to the living environment.RNA editing proteins mainly complete specific base conversion by site-specific deamination of the substrate RNA.Understanding the relationship between the structure and function of RNA editing proteins will facilitate understanding the process of RNA editing and provide a reference for designing artificial editing proteins.PPR protein is a type of RNA editing protein widely distributed in eukaryotes.The DYW subclass is the main type of RNA editing protein in plant mitochondria and chloroplasts.At present,the interaction mechanism between PPR protein and RNA substrate is unclear due to the great difficulty in the purification of wild-type PPR protein.Existing work mainly focused on the recognition function of the N-terminal PPR motif,while a great deficiency was presented in the Cterminal-related research.Except for the DYW structure,which has a deaminase-like structure,the functions of the E and E+ structures remain unclear.OTP82 and CRR22 are RNA editing proteins in arabidopsis,belonging to the DYW subclass.In this project,the function of OTP82 and CRR22 C-terminal fragments were compared.The purification of OTP82 and CRR22 C-terminal fragments was accomplished by establishing a prokaryotic expression system.The relationship between the protein and RNA substrate was detected by gel retardation electrophoresis to reveal the possible mechanism of the C-terminal structure of the PPR DYW subclass.In addition,this project provided basic experimental data for obtaining high-purity wild-type OTP82 by optimizing the conditions of the protein expression and purification in vitro.Two main conclusions were obtained.(1)Although OTP82 and CRR22 belong to the same subclass of PPR protein,the function of their C-terminal E+DYW structures is different.The former prefer to bind the special conformation of the RNA substrate;while the latter form the protein dimers instead of binding RNA substrate.(2)The wild-type OTP82 has 823 amino acids with 13 PPR motifs.The most of expression product of wild-type is in the form of inclusion bodies in E.coli.A certain amount of wild-type OTP82 can be obtained after optimization of denaturation and renaturation conditions.This project gives the clues about the mechanism of RNA editing protein and lays a foundation for the engineering protein design about the PPR proteins.