The Structural Biology Study Of MORF2 Protein And Mitochondrial RNA Polymerase In Mitochondrial In Arabidopsis Thaliana

RNA editing is a post-transcriptional process that alters the genetic information on RNA molecules by insertion,deletion or substitution of bases.RNA editing usually occurs in the plastids and mitochondria of plants,alters the encoded nucleotides,introduces new promoters and terminators,and has biological significance for correcting and regulating translation.Many RNA editing factors have been identified to date.In 2005,the first RNA editing factor,CRR4,was discovered,which is a member of the PLS-type pentapeptide repeat(PPR)family of proteins.It was subsequently discovered that many other PLS-type PPR proteins are involved in plant RNA editing.In fact,all studied PPR proteins are located in plastids or mitochondria.PPR proteins are generally considered to be a class of trans-acting factors involved in RNA editing and function as RNA-binding proteins.There is also a class of non-PPR proteins that are required for RNA editing in chloroplasts and mitochondria of flowering plants.They are called MORF proteins or RIP protein families.The MORF protein is a small protein family with 10 members in Arabidopsis.Among them,MORF2 and MORF9 are located in plastids.MORF1 and MORF3-7 are located in mitochondria.MORF8 is located in both organelles and MORF 10 is considered not involved in RNA editing.Although MORF9 is present in chloroplasts and MORF6 is present in mitochondria,interactions between MORF6 and MORF9 are predicted in the Arabidopsis interaction database.Through yeast double-hybrid experiments and pull down experiments,it is shown that there are interactions between MORF proteins,which can form homologous or heterologous polymers,indicating that MORF proteins may need to work together to participate in editing.Here,we obtained a soluble protein with good status of MORF2 in Arabidopsis thaliana by cloning,expression and purification.The structure of the Arabidopsis chloroplast MORF2 2.4 A resolution was obtained by gas phase diffusion method.Analysis of the structure revealed that MORF2 is a dimer in the crystal.MORF2 uses a typical MORF-box folded structure observed in mitochondrial MORF1 and chloroplast MORF9,showing a similar MORF1 dimer pattern.The difference between MORF2 and MORF9 is attributable to F157(corresponding F162 in MORF1 and W160 in MORF9),which causes a 60° shift in dimerization.The MORF protein contains a characteristic MORF domain that both forms a homologous or heterodimeric molecule and interacts with the RNA editing factor PPR protein and a protein containing the RRM domain.Structural analysis of MORF1,MORF2 and MORF9 indicated that their hydrophobic interface is the basis for the formation of homologous or heterodimers.In the MORF1 structure,a disulfide bond between two C85 residues bridges two dimers and forms a tetramer.However,this absolutely conserved cysteine in all MORF proteins did not work in MORF2 and MORF9 and could not form tetramers.The structure of the(PLS)3PPR-MORF9 complex showed that each PLS binds to one MORF9 monomer,indicating that the dimer of the MORF protein dissociates during the formation of the RNA editor.Structural analysis of the MORF2 dimer and the mode of PPR-MORF2 complex revealed that the interface of the(PLS)3PPR-MORF2 complex overlaps with the dimerization interface.RNA polymerase is a biocatalyst necessary for the synthesis of RNA using DNA as a template.Most prokaryotic RNA polymerases are composed of a core enzyme and a transcription initiation factor sigma factor,which constitute a holoenzyme with a molecular mass of about 500 kDa polymerase.Multi-subunit RNA polymerases are also present in eukaryotes,which are more complex than prokaryotes,namely RNAP ?,RNAP ? and RNAP ?.Some phage RNA polymerases,such as T7,T3,SP6 and K11,and mitochondrial RNA polymerase are single-subunit polymerases compared to prokaryotic and eukaryotic RNA polymerases.In angiosperms,bacterial RNA polymerase(called PEP of plastid-encoded plastid RNA polymerase)is insufficient to transcribe chloroplast genes,requiring one or two phage-type RNA polymerases(called nuclear-encoded plastids).The activity of RNA polymerase(NEP)is supplemented.There are three phage-type RNA polymerases in angiosperms,which are located in the plastid(RPOTp),mitochondrial(RPOTm)and both organelles(RPOTmp).In this paper,the second topic of research is the structure and function of Arabidopsis mitochondrial RNA polymerase.In the research of this subject,a plurality of recombinant plasmids were constructed by truncating different sequences and different vectors to obtain a soluble protein with a relatively uniform state.Crystallization by gas phase diffusion method attempts to change the protein concentration at the time of crystallization,the temperature of crystal growth,and different crystal kits,but no protein crystals are currently obtained.Subsequent changes in conditions to explore protein crystallization conditions and diffraction data were collected to elucidate the transcriptional mechanism of mitochondrial RNA polymerase from an atomic perspective.