Molecular mechanisms for transcription in mammalian mitochondria

Sammanfattning: The circular double stranded mitochondrial genome (mtDNA), which is about 16,600 bp in humans and 16,300 bp in mice, encodes 13 of the -90 different proteins present in the respiratory chain of mammalian mitochondria. The remaining components of the respiratory chain are encoded by nuclear genes and imported into the mitochondrial network. The genes present in mtDNA, 13 mRNAs, 22 tRNAs, and 2 rRNAs, are all essential for oxidative phosphorylation. Therefore, mtDNA replication and transcription are necessary processes for normal function of the respiratory chain and for the metabolism of the eukaryotic cell. In this thesis, we characterized the basic mtDNA transcription machinery in mammals. We identified two novel mitochondrial transcription factors, B1 (TFB1M) and B2 (TFB2M) and for the first time we reconstituted mammalian mitochondrial transcription in vitro by using only pure recombinant proteins. We showed that either TFB1M or TFB2M can support promoter-specific mtDNA transcription in vitro if combined with mitochondrial RNA polymerase (POLRMT) and mitochondrial transcription factor A (TFAM). Studies by us and others suggest that TFB1M and TFB2M may have distinct functions: TFB2M may be primarily a transcription factor and TFB1M a methyltransferase. We identified homologues to TFB1M and TFB2M in many metazoans, including Drosophila melanogaster, indicating a duplication event of the TFBM gene early in evolution. We used the recombinant in vitro transcription system to investigate molecular mechanisms for mtDNA promoter recognition in mammals. The transcription machineries reconstituted from mouse and human cells do not recognize the light strand promoter from the other species. By swapping transcription factors between the mouse and the human transcription machineries, we demonstrated that the observed promoter specificity is governed by POLRMT and TFAM. In contrast, TFB2M does not influence the sequence specificity in these two mammalian systems. TFAM is a dual function protein. The protein binds without sequence specificity to the entire mtDNA molecule, thereby contributing to the formation of the nucleoid, a compact mtDNAprotein complex present in the mitochondrial matrix. TFAM is also an essential transcription factor, which interacts sequence specifically with mitochondrial promoter elements. We investigated the functional consequences of TFAM overexpression or TFAM knockdown in HeLa cells. We concluded that the mtDNA amount is directly correlated with the amount of TFAM and not with the levels of mitochondrial transcription. Our findings indicate that TFAM has a function in mtDNA maintenance and copy number control, which is independent of its role as a transcription factor. Finally, we used the human recombinant mitochondrial transcription system to demonstrate that the conserved sequence block II (CSB II) acts as a sequence-dependent transcription termination element in vitro. In mitochondria, transcription generates RNA primers needed for initiation of leading (heavy) DNA strand synthesis. We found that transcription from the light strand promoter is prematurely terminated at CSB II, downstream of the promoter. This premature termination event is critically dependent on the exact CSB II sequence. The 3 ends of the prematurely terminated transcripts coincide with the major points of RNA to DNA transition in leading strand DNA replication. These findings suggest that primer formation is directed by specific mtDNA sequence elements and thereby provide a novel model for initiation of mtDNA replication in mammalian cells.