Molecular Mechanism Of Arabidopsis Chloroplast Protein PRIN2 Regulating Chloroplast Gene Expression

Plastid encoding RNA polymerase(PEP)is composed of numerous subunits which contains nucleous-encoded proteins and the plastid-encoded subunits.PRIN2 protein was encoded by the nuclear genome and localized in chloroplast nucleoid,and it was reported to be involved in regulation of PEP-transcribed plastid genes,and participation in the communication between the nuclear and the plastid.In this dissertation,the molecular mechanism of PRIN2 in the plastid gene expression were investigated.Fluorescent localization experiment demonstrate PRIN2 localized in chloroplast.Furthermore,immuneblotting analysis showed that PRIN2 is associated with thylakoids.q RT-PCR analysis showed that the expression of PEP-dependent photosynthesis genes,such as,psb A and rbc L was significantly reduced in the prin2 mutant,compared with those in wild type.The transcriptional levels of both NEP and PEP dependent plastid genes,16 S r RNA and atp B were also reduced,while the transcriptional levels of NEP-dependent chloroplast gene,rpo B and acc D,respectively,were 1.1 times and 1.2 times of those in wild type.In this mutant,the amount of the two nuclear-encoded photosynthesis related genes,LHCB and Rbc S,were 80% and 85% of those in the wild type.These results demonstrated that PRIN2 were involved in regulating the PEP-responsible chloroplast gene expression.RT-PCR results analysis showed that the loss of the PRIN2 does not affect on the chloroplast transcription splicing.Yeast two hybrid assays showed that PRIN2 interacts with both FSD3 and At Mur E,two essential components of PEP RNA polymerase.BN-PAGE,two-dimensional electrophoresis and immmuneblotting experiments showed that the PRIN2 protein was present in 669 k Da PEP complex,and co-migrated with the core subunit,rpo B,suggesting PRIN2 was present in PEP complex.PRIN2 were also present in 140-232 k Da complex.Interestingly,the distribution pattern of PRIN2 shared with those of FSD3,supporting that their interaction in vivo.Co-Immunoprecipitation analysis showed that PRIN2 interacts with FSD3 in plant.PRIN2 protein contains an unknown domain,DUF1661,which could form alpha helix structure.The recombinant PRIN2 protein was able to bind the DNA fragments of 16 Sr RNA,pet D,atp B,psb C,psb A,psb B genes' promoter region and the psa B gene' transcriptional region,which are transcribed by PEP.In contrast,the recombinant PRIN2 protein is not able to bind the promoter DNA fragment of acc D and rpo A,which are transcribed by NEP.Through sequence alignment,we found that PRIN2 may bind AT-rich sequence.Yeast two-hybrid experiment analysis shows that PRIN2 has physical interaction with At Sig6 in yeast,but not interact with the other? factors,and At Sig6 interacts with PRIN2 protein.Taken together,these results extended our knowledge of the PRIN2 protein in chloroplast transcription in higher plants,and improved our understanding of the chloroplast PEP polymerase transcriptional apparatus in higher plants.