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Gerald S. Shadel - One of the best experts on this subject based on the ideXlab platform.
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Transcription-independent role for human Mitochondrial RNA polymerase in Mitochondrial ribosome biogenesis
Nucleic acids research, 2013Co-Authors: Yulia V. Surovtseva, Gerald S. ShadelAbstract:Human Mitochondrial RNA polymerase, POLRMT, is required for Mitochondrial DNA (mtDNA) transcription and forms initiation complexes with human Mitochondrial transcription factor B2 (h-mtTFB2). However, POLRMT also interacts with the paralogue of h-mtTFB2, h-mtTFB1, which is a 12S ribosomal RNA methyltransferase required for small (28S) Mitochondrial ribosome subunit assembly. Herein, we show that POLRMT associates with h-mtTFB1 in 28S Mitochondrial ribosome complexes that are stable in the absence of Mitochondrial transcription and distinct from transcription complexes containing POLRMT and h-mtTFB2. Overexpression of POLRMT in HeLa cells increases 12S rRNA methylation by h-mtTFB1 and reduces the steady-state levels of mtDNA-encoded proteins and respiration, apparently because of a decrease in fully assembled 55S Mitochondrial ribosomes. We propose that POLRMT interacts directly with h-mtTFB1 in 28S Mitochondrial ribosomes to augment its 12S rRNA methyltransferase activity, and that together they provide a checkpoint for proper 28S and 55S Mitochondrial ribosome assembly. Thus, POLRMT is multi-functional, forming distinct protein complexes that regulate different steps in Mitochondrial gene expression, at least one of which does not involve transcription per se. The significance of these results is discussed with regard to the mechanism and regulation of human Mitochondrial gene expression and the potential multi-functionality of RNA polymerases in general.
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core human Mitochondrial transcription apparatus is a regulated two component system in vitro
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Timothy E Shutt, Maria F Lodeiro, Justin Cotney, Craig E Cameron, Gerald S. ShadelAbstract:The core human Mitochondrial transcription apparatus is currently regarded as an obligate three-component system comprising the bacteriophage T7-related Mitochondrial RNA polymerase, the rRNA methyltransferase-related transcription factor, h-mtTFB2, and the high mobility group box transcription/DNA-packaging factor, h-mtTFA/TFAM. Using a faithful recombinant human Mitochondrial transcription system from Escherichia coli, we demonstrate that specific initiation from the mtDNA promoters, LSP and HSP1, only requires Mitochondrial RNA polymerase and h-mtTFB2 in vitro. When h-mtTFA is added to these basal components, LSP exhibits a much lower threshold for activation and a larger amplitude response than HSP1. In addition, when LSP and HSP1 are together on the same transcription template, h-mtTFA-independent transcription from HSP1 and h-mtTFA-dependent transcription from both promoters is enhanced and a higher concentration of h-mtTFA is required to stimulate HSP1. Promoter competition experiments revealed that, in addition to LSP competing transcription components away from HSP1, additional cis-acting signals are involved in these aspects of promoter regulation. Based on these results, we speculate that the human Mitochondrial transcription system may have evolved to differentially regulate transcription initiation and transcription-primed mtDNA replication in response to the amount of h-mtTFA associated with nucleoids, which could begin to explain the heterogeneity of nucleoid structure and activity in vivo. Furthermore, this study sheds new light on the evolution of Mitochondrial transcription components by showing that the human system is a regulated two-component system in vitro, and thus more akin to that of budding yeast than thought previously.
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human Mitochondrial ribosomal protein mrpl12 interacts directly with Mitochondrial RNA polymerase to modulate Mitochondrial gene expression
Journal of Biological Chemistry, 2007Co-Authors: Zhibo Wang, Justin Cotney, Gerald S. ShadelAbstract:The core human Mitochondrial transcription machinery comprises a single subunit bacteriophage-related RNA polymerase, POLRMT, the high mobility group box DNA-binding protein h-mtTFA/TFAM, and two transcriptional co-activator proteins, h-mtTFB1 and h-mtTFB2 that also have rRNA methyltransferase activity. Recapitulation of specific initiation of transcription in vitro can be achieved by a complex of POL-RMT, h-mtTFA, and either h-mtTFB1 or h-mtTFB2. However, the nature of Mitochondrial transcription complexes in vivo and the potential involvement of additional proteins in the transcription process in human mitochondria have not been extensively investigated. In Saccharomyces cerevisiae, transcription and translation are physically coupled via the formation of a multiprotein complex nucleated by the binding of Nam1p to the amino-terminal domain of mtRNA polymerase (Rpo41p). This model system paradigm led us to search for proteins that interact with POLRMT to regulate Mitochondrial gene expression in humans. Using an affinity capture strategy to identify POL-RMT-binding proteins, we identified Mitochondrial ribosomal protein L7/L12 (MRPL12) as a protein in HeLa Mitochondrial extracts that interacts specifically with POLRMT in vitro. Purified recombinant MRPL12 binds to POLRMT and stimulates Mitochondrial transcription activity in vitro, demonstrating that this interaction is both direct and functional. Finally, from HeLa cells that overexpress FLAG epitope-tagged MRPL12, increased steady-state levels of mtDNA-encoded transcripts are observed and MRPL12-POLRMT complexes can be co-immunoprecipitated, providing strong evidence that this interaction enhances Mitochondrial transcription or RNA stability in vivo. We speculate that the MRPL12 interaction with POLRMT is likely part of a novel regulatory mechanism that coordinates Mitochondrial transcription with translation and/or ribosome biogenesis during human Mitochondrial gene expression.
Claes M Gustafsson - One of the best experts on this subject based on the ideXlab platform.
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the amino terminal extension of mammalian Mitochondrial RNA polymerase ensures promoter specific transcription initiation
Nucleic Acids Research, 2014Co-Authors: Viktor Posse, Nilsgoran Larsson, Emily Hoberg, Anke Dierckx, Saba Shahzad, Camilla Koolmeister, Marcus L Wilhelmsson, Martin B Hallberg, Claes M GustafssonAbstract:Mammalian Mitochondrial transcription is executed by a single subunit Mitochondrial RNA polymerase (Polrmt) and its two accessory factors, Mitochondrial transcription factors A and B2 (Tfam and Tfb2m). Polrmt is structurally related to single-subunit phage RNA polymerases, but it also contains a unique N-terminal extension (NTE) of unknown function. We here demonstrate that the NTE functions together with Tfam to ensure promoter-specific transcription. When the NTE is deleted, Polrmt can initiate transcription in the absence of Tfam, both from promoters and non-specific DNA sequences. Additionally, when in presence of Tfam and a Mitochondrial promoter, the NTE-deleted mutant has an even higher transcription activity than wild-type polymerase, indicating that the NTE functions as an inhibitory domain. Our studies lead to a model according to which Tfam specifically recruits wild-type Polrmt to promoter sequences, relieving the inhibitory effect of the NTE, as a first step in transcription initiation. In the second step, Tfb2m is recruited into the complex and transcription is initiated.
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Mitochondrial RNA polymerase is needed for activation of the origin of light strand dna replication
Molecular Cell, 2010Co-Authors: Javier Miralles Fuste, Sjoerd Wanrooij, Claes M Gustafsson, Elisabeth Jemt, Caroline Granycome, Tricia J Cluett, Neli Atanassova, Ian J Holt, Maria FalkenbergAbstract:Summary Mitochondrial DNA is replicated by a unique enzymatic machinery, which is distinct from the replication apparatus used for copying the nuclear genome. We examine here the mechanisms of origin-specific initiation of lagging-strand DNA synthesis in human mitochondria. We demonstrate that the Mitochondrial RNA polymerase (POLRMT) is the primase required for initiation of DNA synthesis from the light-strand origin of DNA replication (OriL). Using only purified POLRMT and DNA replication factors, we can faithfully reconstitute OriL-dependent initiation in vitro. Leading-strand DNA synthesis is initiated from the heavy-strand origin of DNA replication and passes OriL. The single-stranded OriL is exposed and adopts a stem-loop structure. At this stage, POLRMT initiates primer synthesis from a poly-dT stretch in the single-stranded loop region. After about 25 nt, POLRMT is replaced by DNA polymerase γ, and DNA synthesis commences. Our findings demonstrate that POLRMT can function as an origin-specific primase in mammalian mitochondria.
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human Mitochondrial RNA polymerase primes lagging strand dna synthesis in vitro
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Sjoerd Wanrooij, Javier Miralles Fuste, Geraldine Farge, Claes M Gustafsson, Maria FalkenbergAbstract:The Mitochondrial transcription machinery synthesizes the RNA primers required for initiation of leading-strand DNA synthesis in mammalian mitochondria. RNA primers are also required for initiation of lagging-strand DNA synthesis, but the responsible enzyme has so far remained elusive. Here, we present a series of observations that suggests that Mitochondrial RNA polymerase (POLRMT) can act as lagging-strand primase in mammalian cells. POLRMT is highly processive on double-stranded DNA, but synthesizes RNA primers with a length of 25 to 75 nt on a single-stranded template. The short RNA primers synthesized by POLRMT are used by the Mitochondrial DNA polymerase γ to initiate DNA synthesis in vitro. Addition of Mitochondrial single-stranded DNA binding protein (mtSSB) reduces overall levels of primer synthesis, but stimulates primer-dependent DNA synthesis. Furthermore, when combined, POLRMT, DNA polymerase γ, the DNA helicase TWINKLE, and mtSSB are capable of simultaneous leading- and lagging-strand DNA synthesis in vitro. Based on our observations, we suggest that POLRMT is the lagging-strand primase in mammalian mitochondria.
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the Mitochondrial RNA polymerase contributes critically to promoter specificity in mammalian cells
The EMBO Journal, 2004Co-Authors: Martina Gaspari, Maria Falkenberg, Nilsgoran Larsson, Claes M GustafssonAbstract:Initiation of transcription in mammalian mitochondria depends on three proteins: Mitochondrial RNA polymerase (POLRMT), Mitochondrial transcription factor A (TFAM) and Mitochondrial transcription factor B2 (TFB2M). We show here that the recombinant mouse and human transcription machineries are unable to initiate transcription in vitro from the heterologous light-strand promoter (LSP) of Mitochondrial DNA. This species specificity is dependent on the interaction of TFAM and POLRMT with specific distal and proximal promoter elements. A sequence element localized from position −1 to −2 relative to the transcription start site in LSP functionally interacts with POLRMT. The POLRMT/TFB2M heterodimer is unable to interact with promoter elements and initiate even abortive transcription in the absence of TFAM. TFAM is thus an integral part of the mammalian transcription machinery, and we propose that TFAM induces a structural change of the promoter that is required for POLRMT-dependent promoter recognition.
Alexis A. Jourdain - One of the best experts on this subject based on the ideXlab platform.
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the pseudouridine synthase rpusd4 is an essential component of Mitochondrial RNA granules
Journal of Biological Chemistry, 2017Co-Authors: Sofia Zaganelli, Alexis A. Jourdain, Kinsey Maundrell, Agata Rozanska, Pedro Rebeloguiomar, Sandra Pierredon, Christopher A Powell, Nicolas Hulo, Robert N LightowlersAbstract:Mitochondrial gene expression is a fundamental process that is largely dependent on nuclear-encoded proteins. Several steps of Mitochondrial RNA processing and maturation, including RNA post-transcriptional modification, appear to be spatially organized into distinct foci, which we have previously termed Mitochondrial RNA granules (MRGs). Although an increasing number of proteins have been localized to MRGs, a comprehensive analysis of the proteome of these structures is still lacking. Here, we have applied a microscopy-based approach that has allowed us to identify novel components of the MRG proteome. Among these, we have focused our attention on RPUSD4, an uncharacterized Mitochondrial putative pseudouridine synthase. We show that RPUSD4 depletion leads to a severe reduction of the steady-state level of the 16S Mitochondrial (mt) rRNA with defects in the biogenesis of the mitoribosome large subunit and consequently in Mitochondrial translation. We report that RPUSD4 binds 16S mt-rRNA, mt-tRNAMet, and mt-tRNAPhe, and we demonstrate that it is responsible for pseudouridylation of the latter. These data provide new insights into the relevance of RNA pseudouridylation in Mitochondrial gene expression.
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Mitochondrial RNA granules: Compartmentalizing Mitochondrial gene expression
Journal of Cell Biology, 2016Co-Authors: Alexis A. Jourdain, Erik Boehm, Kinsey Maundrell, Jeanclaude MartinouAbstract:In mitochondria, DNA replication, gene expression, and RNA degradation machineries coexist within a common nondelimited space, raising the question of how functional compartmentalization of gene expression is achieved. Here, we discuss the recently characterized “Mitochondrial RNA granules,” Mitochondrial subdomains with an emerging role in the regulation of gene expression.
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a mitochondria specific isoform of fastk is present in Mitochondrial RNA granules and regulates gene expression and function
Cell Reports, 2015Co-Authors: Alexis A. Jourdain, Kinsey Maundrell, Mirko Koppen, Chris D Rodley, Naig Gueguen, Pascal Reynier, Adela Guaras, Jose Antonio Enriquez, Paul Anderson, Maria SimarroAbstract:The Mitochondrial genome relies heavily on post-transcriptional events for its proper expression, and misregulation of this process can cause Mitochondrial genetic diseases in humans. Here, we report that a novel translational variant of Fas-activated serine/threonine kinase (FASTK) co-localizes with Mitochondrial RNA granules and is required for the biogenesis of ND6 mRNA, a Mitochondrial-encoded subunit of the NADH dehydrogenase complex (complex I). We show that ablating FASTK expression in cultured cells and mice results specifically in loss of ND6 mRNA and reduced complex I activity in vivo. FASTK binds at multiple sites along the ND6 mRNA and its precursors and cooperates with the Mitochondrial degradosome to ensure regulated ND6 mRNA biogenesis. These data provide insights into the mechanism and control of Mitochondrial RNA processing within Mitochondrial RNA granules.
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grsf1 regulates RNA processing in Mitochondrial RNA granules
Cell Metabolism, 2013Co-Authors: Alexis A. Jourdain, Mateusz Wydro, Zofia M A Chrzanowskalightowlers, Robert N Lightowlers, Mirko Koppen, Chris D Rodley, Jeanclaude MartinouAbstract:Various specialized domains have been described in the cytosol and the nucleus; however, little is known about compartmentalization within the Mitochondrial matrix. GRSF1 (G-rich sequence factor 1) is an RNA binding protein that was previously reported to localize in the cytosol. We found that an isoform of GRSF1 accumulates in discrete foci in the Mitochondrial matrix. These foci are composed of nascent Mitochondrial RNA and also contain RNAse P, an enzyme that participates in Mitochondrial RNA processing. GRSF1 was found to interact with RNAse P and to be required for processing of both classical and tRNA-less RNA precursors. In its absence, cleavage of primary RNA transcripts is abnormal, leading to decreased expression of Mitochondrially encoded proteins and Mitochondrial dysfunction. Our findings suggest that the foci containing GRSF1 and RNAse P correspond to sites where primary RNA transcripts converge to be processed. We have termed these large ribonucleoprotein structures "Mitochondrial RNA granules."
Smita S. Patel - One of the best experts on this subject based on the ideXlab platform.
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Assembly and Cryo-EM structure determination of yeast Mitochondrial RNA polymerase initiation complex intermediates
'Elsevier BV', 2021Co-Authors: Sergio E. Martinez, Brent De Wijngaert, Shemaila Sultana, Chhaya Dharia, Hans Vanbuel, Jiayu Shen, Daniel Vasilchuk, Smita S. Patel, Anupam Singh, Kalyan DasAbstract:Summary: In yeast mitochondria, transcription initiation requires assembly of Mitochondrial RNA polymerase and transcription initiation factor MTF1 at the DNA promoter initiation site. This protocol describes the purification of the component proteins and assembly of partially melted and fully melted initiation complex states. Both states co-exist in equilibrium in the same sample as seen by cryoelectron microscopy (cryo-EM) and allow elucidation of MTF1’s structural roles in controlling the transition into elongation. We further outline how analysis of the complex by light scattering, thermal shift assay, and ultrafiltration assay exhibits reproducible results.For complete details on the use and execution of this protocol, please refer to De Wijngaert et al. (2021)
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cryo em structures reveal transcription initiation steps by yeast Mitochondrial RNA polymerase
Social Science Research Network, 2020Co-Authors: Brent De Wijngaert, Shemaila Sultana, Chhaya Dharia, Hans Vanbuel, Jiayu Shen, Daniel Vasilchuk, Sergio E. Martinez, Eaazhisai Kandiah, Smita S. Patel, Kalyan DasAbstract:Understanding of Mitochondrial DNA transcription initiation lags that of bacterial and nuclear DNA transcription. We have solved the structures of yeast Mitochondrial RNA polymerase transcription pre-initiation complex (PIC) and initiation complex (IC) with fully resolved transcription bubbles. The structures explain promoter melting, template alignment, DNA scrunching, transition into elongation, and abortive synthesis. Partially melted DNA in PIC is caused by interactions of MTF1 with four flipped non-template bases. In IC, the bubble expands, a largescale movement aligns the template with RNA/NTP in the active site, and the downstream DNA is repositioned. The IC-state captures the scrunched DNA as an NT-loop that interacts with the centrally positioned MTF1 C-tail. Coexisting scrunched and unscrunched states and imminent steric clashes of NT-loop and RNA:DNA with C-tail explain abortive synthesis and transition into elongation. The IC-state with UTPαS poised for catalytic incorporation provides a platform to assess the toxicity of antiviral nucleosides/nucleotides like remdesivir.
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Cryo-EM structures reveal transcription initiation steps by yeast Mitochondrial RNA polymerase
2020Co-Authors: Brent De Wijngaert, Shemaila Sultana, Chhaya Dharia, Hans Vanbuel, Jiayu Shen, Daniel Vasilchuk, Sergio E. Martinez, Eaazhisai Kandiah, Smita S. Patel, Kalyan DasAbstract:Cryo-EM structures of transcription pre-initiation complex (PIC) and initiation complex (IC) of yeast Mitochondrial RNA polymerase show fully resolved transcription bubbles and explain promoter melting, template alignment, DNA scrunching, transition into elongation, and abortive synthesis. Promoter melting initiates in PIC with MTF1 trapping the -4 to -2 non-template (NT) bases in its NT-groove. Transition to IC is marked by a large-scale movement that aligns the template with RNA at the active site. RNA synthesis scrunches the NT strand into an NT-loop, which interacts with centrally positioned MTF1 C-tail. Steric clashes of the C-tail with RNA:DNA and NT-loop, and dynamic scrunching-unscrunching of DNA explain abortive synthesis and transition into elongation. Capturing the catalytically active IC-state with UTPαS poised for incorporation enables modeling toxicity of antiviral nucleosides/nucleotides.
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Mitochondrial RNA capping highly efficient 5 RNA capping with nad and nadh by yeast and human Mitochondrial RNA polymerase
bioRxiv, 2018Co-Authors: Jeremy G Bird, Dmitry Temiakov, Smita S. Patel, Aparna Ramachandran, Urmimala Basu, David Kuster, Ewa Grudziennogalska, Megerditch Kiledjian, Richard H EbrightAbstract:Bacterial and eukaryotic nuclear RNA polymerases (RNAPs) cap RNA with the oxidized and reduced forms of the metabolic effector nicotinamide adenine dinucleotide, NAD + and NADH, using NAD + and NADH as non canonical initiating nucleotides for transcription initiation. Here, we show that Mitochondrial RNAPs (mtRNAPs) cap RNA with NAD + and NADH, and do so more efficiently than nuclear RNAPs. Direct quantitation of NAD + - and NADH-capped RNA demonstrates remarkably high levels of capping in vivo: up to ~50% NAD + and ~40% NADH capping of yeast Mitochondrial transcripts, and up to ~10% NAD + capping of human Mitochondrial transcripts. The capping efficiency is determined by promoter sequence at and upstream of the transcription start site and, in yeast and human cells, by intracellular NAD + and NADH levels. Our findings indicate mtRNAPs serve as both sensors and actuators in coupling cellular metabolism to Mitochondrial gene expression, sensing NAD + and NADH levels and adjusting transcriptional outputs accordingly.
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the yeast Mitochondrial RNA polymerase and transcription factor complex catalyzes efficient priming of dna synthesis on single stranded dna
Journal of Biological Chemistry, 2016Co-Authors: Aparna Ramachandran, Aishwarya P Deshpande, Divya Nandakumar, Thomas P Lucas, Ramanagouda Rbhojappa, Guoqing Tang, Kevin D Raney, Whitney Y Yin, Smita S. PatelAbstract:Abstract Primases use single-stranded (ss) DNAs as templates to synthesize short oligoribonucleotide primers that initiate lagging strand DNA synthesis or reprime DNA synthesis after replication fork collapse, but the origin of this activity in the mitochondria remains unclear. Herein, we show that the Saccharomyces cerevisiae Mitochondrial RNA polymerase (Rpo41) and its transcription factor (Mtf1) is an efficient primase that initiates DNA synthesis on ssDNA coated with the yeast Mitochondrial ssDNA-binding protein, Rim1. Both Rpo41 and Rpo41-Mtf1 can synthesize short and long RNAs on ssDNA template and prime DNA synthesis by the yeast Mitochondrial DNA polymerase Mip1. However, the ssDNA-binding protein Rim1 severely inhibits the RNA synthesis activity of Rpo41, but not the Rpo41-Mtf1 complex, which continues to prime DNA synthesis efficiently in the presence of Rim1. We show that RNAs as short as 10–12 nt serve as primers for DNA synthesis. Characterization of the RNA-DNA products shows that Rpo41 and Rpo41-Mtf1 have slightly different priming specificity. However, both prefer to initiate with ATP from short priming sequences such as 3′-TCC, TTC, and TTT, and the consensus sequence is 3′-Pu(Py)2–3. Based on our studies, we propose that Rpo41-Mtf1 is an attractive candidate for serving as the primase to initiate lagging strand DNA synthesis during normal replication and/or to restart stalled replication from downstream ssDNA.
Maria Falkenberg - One of the best experts on this subject based on the ideXlab platform.
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Mitochondrial RNA polymerase is needed for activation of the origin of light strand dna replication
Molecular Cell, 2010Co-Authors: Javier Miralles Fuste, Sjoerd Wanrooij, Claes M Gustafsson, Elisabeth Jemt, Caroline Granycome, Tricia J Cluett, Neli Atanassova, Ian J Holt, Maria FalkenbergAbstract:Summary Mitochondrial DNA is replicated by a unique enzymatic machinery, which is distinct from the replication apparatus used for copying the nuclear genome. We examine here the mechanisms of origin-specific initiation of lagging-strand DNA synthesis in human mitochondria. We demonstrate that the Mitochondrial RNA polymerase (POLRMT) is the primase required for initiation of DNA synthesis from the light-strand origin of DNA replication (OriL). Using only purified POLRMT and DNA replication factors, we can faithfully reconstitute OriL-dependent initiation in vitro. Leading-strand DNA synthesis is initiated from the heavy-strand origin of DNA replication and passes OriL. The single-stranded OriL is exposed and adopts a stem-loop structure. At this stage, POLRMT initiates primer synthesis from a poly-dT stretch in the single-stranded loop region. After about 25 nt, POLRMT is replaced by DNA polymerase γ, and DNA synthesis commences. Our findings demonstrate that POLRMT can function as an origin-specific primase in mammalian mitochondria.
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human Mitochondrial RNA polymerase primes lagging strand dna synthesis in vitro
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Sjoerd Wanrooij, Javier Miralles Fuste, Geraldine Farge, Claes M Gustafsson, Maria FalkenbergAbstract:The Mitochondrial transcription machinery synthesizes the RNA primers required for initiation of leading-strand DNA synthesis in mammalian mitochondria. RNA primers are also required for initiation of lagging-strand DNA synthesis, but the responsible enzyme has so far remained elusive. Here, we present a series of observations that suggests that Mitochondrial RNA polymerase (POLRMT) can act as lagging-strand primase in mammalian cells. POLRMT is highly processive on double-stranded DNA, but synthesizes RNA primers with a length of 25 to 75 nt on a single-stranded template. The short RNA primers synthesized by POLRMT are used by the Mitochondrial DNA polymerase γ to initiate DNA synthesis in vitro. Addition of Mitochondrial single-stranded DNA binding protein (mtSSB) reduces overall levels of primer synthesis, but stimulates primer-dependent DNA synthesis. Furthermore, when combined, POLRMT, DNA polymerase γ, the DNA helicase TWINKLE, and mtSSB are capable of simultaneous leading- and lagging-strand DNA synthesis in vitro. Based on our observations, we suggest that POLRMT is the lagging-strand primase in mammalian mitochondria.
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the Mitochondrial RNA polymerase contributes critically to promoter specificity in mammalian cells
The EMBO Journal, 2004Co-Authors: Martina Gaspari, Maria Falkenberg, Nilsgoran Larsson, Claes M GustafssonAbstract:Initiation of transcription in mammalian mitochondria depends on three proteins: Mitochondrial RNA polymerase (POLRMT), Mitochondrial transcription factor A (TFAM) and Mitochondrial transcription factor B2 (TFB2M). We show here that the recombinant mouse and human transcription machineries are unable to initiate transcription in vitro from the heterologous light-strand promoter (LSP) of Mitochondrial DNA. This species specificity is dependent on the interaction of TFAM and POLRMT with specific distal and proximal promoter elements. A sequence element localized from position −1 to −2 relative to the transcription start site in LSP functionally interacts with POLRMT. The POLRMT/TFB2M heterodimer is unable to interact with promoter elements and initiate even abortive transcription in the absence of TFAM. TFAM is thus an integral part of the mammalian transcription machinery, and we propose that TFAM induces a structural change of the promoter that is required for POLRMT-dependent promoter recognition.