Structure of human mitochondrial RNA polymerase. Nature

Gene Center and Department of Biochemistry, Center for Integrated Protein Science Munich, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.
Nature (Impact Factor: 41.46). 09/2011; 478(7368):269-73. DOI: 10.1038/nature10435
Source: PubMed


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. Whereas T7 RNAP can initiate transcription by itself, mtRNAP requires the factors TFAM and TFB2M for binding and melting promoter DNA. 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. 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. 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.

11 Reads
  • Source
    • "The central component of the transcription machinery in eukaryotes is the mitochondrial RNA polymerase (POLRMT), believed to have originated from the T-odd phage family as a result of an ancient phage-integration event (8,9). POLRMTs have a conserved 80 kDa C-terminal domain that shares significant structural homology with their T-odd phage counterparts (10). Despite this considerable homology, the mechanism of mitochondrial transcription differs from that in T-odd phages in some important aspects. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Initiation of transcription in human mitochondria involves two factors, TFAM and TFB2M, in addition to the mitochondrial RNA polymerase, POLRMT. We have investigated the organization of the human mitochondrial transcription initiation complex on the light-strand promoter (LSP) through solution X-ray scattering, electron microscopy (EM) and biochemical studies. Our EM results demonstrate a compact organization of the initiation complex, suggesting that protein-protein interactions might help mediate initiation. We demonstrate that, in the absence of DNA, only POLRMT and TFAM form a stable interaction, albeit one with low affinity. This is consistent with the expected transient nature of the interactions necessary for initiation and implies that the promoter DNA acts as a scaffold that enables formation of the full initiation complex. Docking of known crystal structures into our EM maps results in a model for transcriptional initiation that strongly correlates with new and existing biochemical observations. Our results reveal the organization of TFAM, POLRMT and TFB2M around the LSP and represent the first structural characterization of the entire mitochondrial transcriptional initiation complex.
    Nucleic Acids Research 01/2014; 42(6). DOI:10.1093/nar/gkt1360 · 9.11 Impact Factor
  • Source
    • "Interactions between the AT-rich recognition loop and the upstream promoter region are important features of transcription initiation by phage RNAPs (40,41). It is likely that lack of these interactions renders mtRNAP less specific and decreases its affinity to the promoter (2). Tight binding of TFAM to its recognition sequence at a defined position relative to the promoter start site, coupled with its association with mtRNAP, compensate for these changes, and appear to be crucial conditions for de novo RNA synthesis in mammalian mitochondria (Figure 6). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The mitochondrial genome is transcribed by a single-subunit T7 phage-like RNA polymerase (mtRNAP), structurally unrelated to cellular RNAPs. In higher eukaryotes, mtRNAP requires two transcription factors for efficient initiation-TFAM, a major nucleoid protein, and TFB2M, a transient component of mtRNAP catalytic site. The mechanisms behind assembly of the mitochondrial transcription machinery and its regulation are poorly understood. We isolated and identified a previously unknown human mitochondrial transcription intermediate-a pre-initiation complex that includes mtRNAP, TFAM and promoter DNA. Using protein-protein cross-linking, we demonstrate that human TFAM binds to the N-terminal domain of mtRNAP, which results in bending of the promoter DNA around mtRNAP. The subsequent recruitment of TFB2M induces promoter melting and formation of an open initiation complex. Our data indicate that the pre-initiation complex is likely to be an important target for transcription regulation and provide basis for further structural, biochemical and biophysical studies of mitochondrial transcription.
    Nucleic Acids Research 01/2014; 42(6). DOI:10.1093/nar/gkt1356 · 9.11 Impact Factor
  • Source
    • "Alignment of the deduced protein sequence with homologs downloaded from Genbank and JGI showed that it had all nine of the conserved sequence blocks found in the catalytic C-terminal domain of the protein structure [14], [37], [40] but was quite divergent between these blocks (Fig. 2A). There was very little sequence relatedness in the N-terminal domain of the protein, which is largely involved in promoter binding and initiation of transcription [21], [22]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Dinoflagellates are a large group of algae that contribute significantly to marine productivity and are essential photosynthetic symbionts of corals. Although these algae have fully-functioning mitochondria and chloroplasts, both their organelle genomes have been highly reduced and the genes fragmented and rearranged, with many aberrant transcripts. However, nothing is known about their RNA polymerases. We cloned and sequenced the gene for the nuclear-encoded mitochondrial polymerase (RpoTm) of the dinoflagellate Heterocapsa triquetra and showed that the protein presequence targeted a GFP construct into yeast mitochondria. The gene belongs to a small gene family, which includes a variety of 3'-truncated copies that may have originated by retroposition. The catalytic C-terminal domain of the protein shares nine conserved sequence blocks with other single-subunit polymerases and is predicted to have the same fold as the human enzyme. However, the N-terminal (promoter binding/transcription initiation) domain is not well-conserved. In conjunction with the degenerate nature of the mitochondrial genome, this suggests a requirement for novel accessory factors to ensure the accurate production of functional mRNAs.
    PLoS ONE 06/2013; 8(6):e65387. DOI:10.1371/journal.pone.0065387 · 3.23 Impact Factor
Show more