A Research On The Mitochondrial Transcription Mechanism In S.Pombe

As an important organelle, apart from its central role in ATP production, mitochondrion is involved in many physical processes such as metabolism and apoptosis. Mitochondria have their own circular genomes, whose transcription is accomplished by the nuclear encoded mitochondrial RNA polymerases and their transcription factors. The mitochondrial RNA polymerase is a single subunit RNA polymerase (mtRNAP) whose amino acid sequence shares much similarity to that of T7RNA polymerase (T7RNAP). Much of the research on mitochondrial transcription was done based on the system in human cell or budding yeast (S.Cerevisiae). The fission yeast, S.Pombe, a widely used model organism for cell cycle control and differential research, is emerging as an attractive model for studying mitochondrion. Yet, little is known about the exact mechanism of its mitochondrial transcription. Considering the disparity between S.Pombe and S.Cerevisiae in term of mtDNA size and composition, it might be inappropriate in some occasion to extrapolate the mitochondrial transcription mechanism in S.Pombe directly based on that of S.Cerevisiae. Recently, it was found the mitochondrial transcription factor Mtfl is involved in the regulation of a stress related gene srkl in S.Pombe. It seems urgent to gain some exact knowledge about the mechanism of mitochondrial transcription in S.Pombe for the better understanding of the nucleus-mitochondria cross talks like this.Previously, it was found in our lab that the mitochondrial RNA polymerase Rpo41and its transcription factor Mtfl are essential for S.Pombe, both Rpo41and Mtfl are implicated in the transcription of the mt-genome. Based on that, in this work we purified the Rpo41and Mtfl of S.Pombe and tested its transcription activity in vitro in a radio-active free assay. In the in vitro transcription tests, it was found that Rpo41can initiate transcription from linear double stranded DNA in the absence of Mtf1.5’-RACE of the in vitro transcripts confirmed this finding, and the transcription start sites (TSS) in vitro matches the ones of in vivo, both in the presence or in the absence of Mtf1.The interactions among Mtfl, Rpo41and the promoter containing DNA were tested using techniques such as GST Pull-down, SPR, EMSA and Dnasel footprinting. Weak interaction between Mtfl and Rpo41was observed in solution, which was greatly enhanced by the presence of the promoter containing dsDNA. In EMSA experiment, unspecific binding of Mtfl or Rpo41alone to promoter DNA was discernible, while presence of both Mtfl and Rpo41caused a distinct specific super shift. The exact range of Rpo41and Mtfl binding to DNA was mapped to-25～+14 relative to the transcription start site on the coding strand, which was comparably wider than that in Cerevisiae. In addition, it was inferred from these experiments that Mtfl forms dimmer when it works together with Rpo41to bind DNA. Then we tested different DNA template for transcription. Results indicated that the three promoters in S.Pombe differ little both in sequence and transcription efficiency, the divergence is mainly conferred by the promoter flanking sequences. The upstream regions of the promoter have effect on transcription, but this effect is relatively limited. The real sources of transcription efficiency difference among the three promoters originate from the promoter downstream sequences. In accordance with the situation in S.Cerevisiae, at position of+2from TSS, T and C base inhibits transcription greatly. At least25bases downstream the TSS are involved in the transcriptional control. Specially, consecutive Gs downstream the TSS have conspicuous inhibitory action on transcription, which is in proportion to the count of G bases and is in inverse proportion to the distance from the consecutive G to the TSS. It seems the effect of consecutive Gs at+27～+30from TSS is too weak to notice. Furthermore, we found Rpo41and Mtfl can initiate transcription on single stranded DNA specifically from promoter. By using single stranded DNA as probe in Anisotropy and EMSA experiment, it was found (1) Rpo41itself makes little discrimination between single strand DNA and double stranded DNA, while upon the auxiliary of Mtfl, the polymerase and the transcription factor bind preferentially to single strand DNA that carries the promoter;(2) Rpo41and Mtfl complex can recognize the promoter sequence and its downstream sequence on the template strand. Combined with the base content analysis around the promoter sequences, a conjecture can be made that Mtfl accelerated transcription by helping Rpo41to recognize the melted DNA promoter and its downstream sequence on the template strand.Based on these experiments, the scenario of the mitochondrial transcription initiation complex formation can be depicted as such:AT rich promoter region of DNA switches between paired and melted states transiently and frequently due to its thermal dynamic feature. With the help of Mtfl, Rpo41makes brisk capture of the single stranded DNA. When the sequence is recognized, the captured stretch of DNA is scrunched into the deep inside of the polymerase forming the transcription initiation complex, otherwise Rpo41and Mtfl release this stretch of DNA.