SUMMARY Human mitochondrial transcription factor A (TFAM) is a high-mobility group (HMG) protein at the nexus of mitochondrial DNA (mtDNA) replication, transcription and inheritance. Little is known about the mechanisms underlying its post-translational regulation. Here, we demonstrate that TFAM is phosphorylated within its HMG box 1 (HMG1) by cAMP-dependent protein kinase in mitochondria. HMG1 phosphorylation impairs the ability of TFAM to bind DNA and to activate transcription. We show that only DNA-free TFAM is degraded by the Lon protease, which is inhibited by the anti-cancer drug bortezomib. In cells with normal mtDNA levels, HMG1-phosphorylated TFAM is degraded by Lon. However in cells with severe mtDNA deficits, non-phosphorylated TFAM is also degraded as it is DNA-free. Depleting Lon in these cells increases TFAM, and upregulates mtDNA content, albeit transiently. Phosphorylation and proteolysis thus provide mechanisms for rapidly fine-tuning TFAM function and abundance in mitochondria, which are crucial for maintaining and expressing mtDNA.

Transcription of the mitochondrial genome is performed by a single-subunit RNA polymerase (mtRNAP) that is distantly related to the RNAP of bacteriophage T7, the pol I family of DNA polymerases, and single-subunit RNAPs from chloroplasts [1][2][3][4] . Whereas T7 RNAP can initiate transcription by itself, mtRNAP requires the factors TFAM and TFB2M for binding and melting promoter DNA [5][6][7] . TFAM is an abundant protein that binds and bends promoter DNA 15-40 base pairs upstream of the transcription start site, and stimulates the recruitment of mtRNAP and TFB2M to the promoter 8,9 . TFB2M assists mtRNAP in promoter melting and reaches the active site of mtRNAP to interact with the first base pair of the RNA-DNA hybrid 10 . Here we report the X-ray structure of human mtRNAP at 2.5 Å resolution, which reveals a T7-like catalytic carboxy-terminal domain, an amino-terminal domain that remotely resembles the T7 promoter-binding domain, a novel pentatricopeptide repeat domain, and a flexible N-terminal extension. The pentatricopeptide repeat domain sequesters an AT-rich recognition loop, which binds promoter DNA in T7 RNAP, probably explaining the need for TFAM during promoter binding. Consistent with this, substitution of a conserved arginine residue in the AT-rich recognition loop, or release of this loop by deletion of the N-terminal part of mtRNAP, had no effect on transcription. The fingers domain and the intercalating hairpin, which melts DNA in phage RNAPs, are repositioned, explaining the need for TFB2M during promoter melting. Our results provide a new venue for the mechanistic analysis of mitochondrial transcription. They also indicate how an early phage-like mtRNAP lost functions in promoter binding and melting, which were provided by initiation factors in trans during evolution, to enable mitochondrial gene regulation and the adaptation of mitochondrial function to changes in the environment.We crystallized a fully functional variant of a recombinant human mtRNAP (residues 105-1230) that requires the presence of both TFAM and TFB2M for efficient transcription initiation on a doublestranded promoter DNA ( Supplementary Fig. 1). The structure was determined at 2.5 Å resolution by molecular replacement with the use of a truncated T7 RNAP structure as a search model 2 , and was refined to a free R-factor of 0.23 (Methods, and Supplementary Table 1).The mtRNAP structure has the shape of a right hand with palm, fingers and thumb subdomains, characteristic of the pol A family of model with the major domains and structural elements indicated. The CTD that is conserved in all single-stranded RNAPs is in dark grey, the NTD in silver, the PPR domain in blue, and the N-terminal extension helix in sand. The active site is indicated by a magenta sphere for a modelled catalytic metal ion. b, Schematic comparison of mtRNAP with T7 (PDB 1QLN) RNAP. Prominent structural elements are indicated. mtRNAP-specific residues 1-368 include the mitochondrial targeting signal, the N-terminal extension and the PPR domain.Regions in...

Transcription in human mitochondria is driven by a single-subunit, factor-dependent RNA polymerase (mtRNAP). Despite its critical role in both expression and replication of the mitochondrial genome, transcription initiation by mtRNAP remains poorly understood. Here, we report crystal structures of human mitochondrial transcription initiation complexes assembled on both light and heavy strand promoters. The structures reveal how transcription factors TFAM and TFB2M assist mtRNAP to achieve promoter-dependent initiation. TFAM tethers the N-terminal region of mtRNAP to recruit the polymerase to the promoter whereas TFB2M induces structural changes in mtRNAP to enable promoter opening and trapping of the DNA non-template strand. Structural comparisons demonstrate that the initiation mechanism in mitochondria is distinct from that in the well-studied nuclear, bacterial, or bacteriophage transcription systems but that similarities are found on the topological and conceptual level. These results provide a framework for studying the regulation of gene expression and DNA replication in mitochondria.

Coordinated replication and expression of mitochondrial genome is critical for metabolically active cells during various stages of development. However, it is not known whether replication and transcription can occur simultaneously without interfering with each other and whether mtDNA copy number can be regulated by the transcription machinery. We found that interaction of human transcription elongation factor, TEFM with mitochondrial RNA polymerase (mtRNAP) and nascent transcript prevents generation of replication primers and increases transcription processivity thereby serving as a molecular switch between replication and transcription, which appear to be mutually exclusive processes in mitochondria. TEFM may allow mitochondria to increase transcription rates and, as consequence, respiration and ATP production without the need to replicate mtDNA, which has been observed during spermatogenesis and early stages of embryogenesis.

The crystal structure of the human mitochondrial RNA polymerase (mtRNAP) transcription elongation complex was determined at 2.65 Å resolution. The structure reveals a 9–base pair hybrid formed between the DNA template and the RNA transcript and one turn of DNA both upstream and downstream of the hybrid. Comparisons with the distantly related RNAP from bacteriophage T7 indicates conserved mechanisms for substrate binding and nucleotide incorporation, but also strong mechanistic differences. Whereas T7 RNAP refolds during the transition from initiation to elongation, mtRNAP adopts an intermediary conformation that is capable of elongation without refolding. The intercalating hairpin that melts DNA during T7 RNAP initiation separates RNA from DNA during mtRNAP elongation. Newly synthesized RNA exits towards the PPR domain, a unique feature of mtRNAP with conserved RNA recognition motifs.