mTERF8 can Terminate Transcription of the psbJ Gene in vitro

One-sentence Summary: mTERF8 functions as a transcription terminator of chloroplast gene psbJ in Arabidopsis. ABSTRACT 41 Members of the mitochondrial transcription terminator factor (mTERF) family, originally identified 42 in vertebrate mitochondria, are involved in the termination of organellular transcription. In plants, 43 mTERF proteins are mainly localized in chloroplasts and mitochondria. In Arabidopsis thaliana , mTERF8/pTAC15 was identified in the plastid-encoded RNA polymerase (PEP) complex, the 45 major RNA polymerase of chloroplasts. In this work, we demonstrate that mTERF8 is associated 46 with the PEP complex. An mTERF8 knockout line displayed a wild type-like phenotype under 47 standard growth conditions, but showed impaired efficiency of photosystem II electron flow. 48 Transcription of most chloroplast genes was not substantially affected in the mterf8 mutant; 49 however, the level of the psbJ transcript from the psbEFLJ polycistron was increased. RNA blot 50 analysis showed that a larger transcript accumulates in mterf8 than in the wild type. Thus, 51 abnormal transcription and/or RNA processing occur for the psbEFLJ polycistron. Circular 52 reverse transcription polymerase chain reaction and sequence analysis showed that the psbJ 53 transcript terminates 95 nucleotides downstream of the translation stop codon in the wild type, 54 whereas its termination is aberrant in mterf8 . Both electrophoresis mobility shift assays and 55 chloroplast chromatin immunoprecipitation analysis showed that mTERF8 specifically binds to 56 the 3′ terminal region of psbJ . Transcription analysis using the in vitro T7 RNA polymerase 57 system showed that mTERF8 terminates psbJ transcription. Together, these results suggest that 58 mTERF8 is specifically involved in the transcription termination of the chloroplast gene psbJ . in chloroplast nucleoids, co-fractionates with the core polymerase subunit RpoB. An mterf8 is in plastid gene of photosystem II electron The recombinant protein for These suggest that transcription termination specifically binding to the 3′-terminal end of the chloroplast Arabidopsis. light intensity dependence of three chlorophyll fluorescence parameters: light-response curves of non-photochemical quenching (NPQ), PSII quantum yield, photochemical quenching, and electron transport rate. Non-photochemical quenching (NPQ), PSII quantum yield, photochemical quenching, and electron transport rate were slightly lower in the mterf8 mutants than in the wild type ( Figure 2D ). These data indicate a slightly decreased efficiency of photosynthetic electron flow in the mterf8 mutant. We then investigated whether the absence of mTERF8 affects the accumulation of photosynthetic complexes. Thylakoid membranes were solubilized with 2% DM and membrane protein complexes were separated by BN-PAGE. Our results show that the amount of PSII super-complexes is slightly decreased in the mterf8 mutant, compared with wild type ( Supplemental Figure S2 A and B ). Accumulation of the PSII proteins PsbA and PsbB was slightly reduced ( Supplemental Figure S2C ). The other photosynthetic proteins PsaA, PsaD, AtpA, AtpC, CytF, and NdhO accumulated to a similar level as in wild type. extremities the genomic psbJ fragment containing the binding site of mTERF8 pGEM linearization plasmid, MBP:mTERF8 chloroplast psbA rbcL psaJ petD petA their down-stream pGEM plasmid. They linearized T7 MBP:mTERF8 These suggest the 3′ terminal region of psbJ