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...

Summary Transcription in human mitochondria is carried out by a single-subunit, T7-like RNA polymerase assisted by several auxiliary factors. We demonstrate that an essential initiation factor, TFB2, forms a network of interactions with DNA near the transcription start site and facilitates promoter melting but may not be essential for promoter recognition. Unexpectedly, catalytic autolabeling reveals that TFB2 interacts with the priming substrate, suggesting that TFB2 acts as a transient component of the catalytic site of the initiation complex. Mapping of TFB2 identifies a region of its N-terminal domain that is involved in simultaneous interactions with the priming substrate and the templating (+1) DNA base. Our data indicate that the transcriptional machinery in human mitochondria has evolved into a system that combines features inherited from self-sufficient, T7-like RNA polymerase and those typically found in systems comprising cellular multi-subunit polymerases, and provide insights into the molecular mechanisms of transcription regulation in mitochondria.

Human mitochondrial transcription is driven by a single subunit RNA polymerase and a set of basal transcription factors. The development of a recombinant in vitro transcription system has allowed for a detailed molecular characterization of the individual components and their contribution to transcription initiation. We found that TFAM and TFB2M act synergistically and increase transcription efficiency 100 -200-fold as compared with RNA polymerase alone. Both the light-strand promoter (LSP) and the HSP1 promoters displayed maximal levels of in vitro transcription when TFAM was present in an amount equimolar to the DNA template. Importantly, we did not detect any significant transcription activity in the presence of the TFB2M paralog, TFB1M, or when templates containing the putative HSP2 promoter were used. These data confirm previous observations that TFB1M does not function as a bona fide transcription factor and raise questions as to whether HSP2 serves as a functional promoter in vivo. In addition, we did not detect transcription stimulation by the ribosomal protein MRPL12. Thus, only two essential initiation factors, TFAM and TFB2M, and two promoters, LSP and HSP1, are required to drive transcription of the mitochondrial genome.Transcription of the human mitochondrial genome is governed by a nuclear-encoded single-subunit RNA polymerase (POLRMT) that is assisted by two transcription initiation factors, TFAM and TFB2M (see Refs. 1 and 2 and references therein). POLRMT possesses promoter recognition functions but depends on TFAM and TFB2M for promoter melting (3). TFAM, a high mobility group class protein, binds to mitochondrial DNA, protects a region 14 -35 bp upstream of the lightstrand promoter (LSP) 4 transcription start site, and assists in assembly of the initiation complex by attracting POLRMT-TFB2M and/or by causing initial melting of the promoter (4). The primary role of TFB2M is to melt the promoter and to stabilize the open promoter complex by simultaneous binding of the priming substrate and the templating DNA base (5). Although the basic requirements for mitochondrial transcription have been established, a number of existing controversial observations preclude a comprehensive view of gene transcription and its regulation in mitochondria. For example, in addition to TFB2M, human mitochondria also contain a homologous factor TFB1M that was reported to stimulate transcription initiation in vitro with 10 -100-fold lower efficiency (6, 7). However, in vivo studies demonstrated that although TFB1M is an essential methyltransferase required to methylate 12 S ribosomal RNA, it plays no role in transcription (8).Another paradox in the field of mitochondrial transcription concerns the existence of an additional promoter in the heavy strand of mtDNA. Transcription initiated at the LSP results in synthesis of a single mRNA that encodes subunit 6 of the NADH dehydrogenase and eight tRNAs (9). The heavy strand of mtDNA encodes 12 polypeptides, two rRNAs, and the rest of the tRNAs; transcription of this strand...

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