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Proc Natl Acad Sci U S A. 2010 Jul 6; 107(27): 12133–12138.
Published online 2010 Jun 18. doi:  10.1073/pnas.0910581107
PMCID: PMC2901451

Core human mitochondrial transcription apparatus is a regulated two-component system in vitro


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.

Keywords: h-mtTFA/TFAM, mtDNA, nucleoid, POLRMT, h-mtTFB2/TFB2M

Human mtDNA encodes 37 essential genes required for oxidative phosphorylation, mutations in which cause maternally inherited diseases and are also thought to contribute to other more common disorders, aging, and age-related pathology (1, 2). Expression and replication of mtDNA is completely dependent on the nucleus, because all of the regulatory factors required are encoded by nuclear genes and imported into the organelle. For example, mitochondrial gene expression alone involves > 100 nuclear genes, including a dedicated mitochondrial RNA polymerase and associated transcription factors, RNA processing machinery, and the large cadre of proteins involved in mitochondrial translation.

Human mtDNA is an approximately 16.5 kb double-stranded, circular molecule that contains a major noncoding region, called the D-loop regulatory region, which harbors the known promoters for transcription. Transcripts corresponding to both strands of mtDNA are initiated from three promoters designated as the light-strand promoter (LSP) and the heavy-strand promoters 1 and 2 (HSP1 and HSP2) (3). Transcription from the LSP and HSP2 result in long polycistronic products, whereas transcription from HSP1 produces primarily a truncated transcript encoding only the two rRNA species (12S and 16S) and two tRNAs (4). The two rRNAs assemble with nucleus-encoded mitochondrial ribosomal proteins to generate mitochondrial ribosomes. Finally, transcripts from the LSP are also used as primers for mtDNA replication (5, 6), thus LSP transcription serves a dual role in gene expression and mtDNA maintenance (7, 8).

Much effort has been devoted to identifying the core machinery needed for mitochondrial transcription in humans as a critical step toward understanding the mechanism of human mitochondrial gene expression and replication in vivo and its role in human disease. The human mitochondrial RNA polymerase (POLRMT) is a single-subunit enzyme related to the T7 family of bacteriophage RNA polymerases (9, 10). However, unlike T7 RNA polymerase, which does not require any transcription factors, efficient promoter-specific initiation by human POLRMT in vitro requires the high mobility group box transcription factor h-mtTFA/TFAM (referred to as h-mtTFA from this point forward) and one of two rRNA methyltransferase-related transcription factors, h-mtTFB1 and h-mtTFB2 (1114). Based on work in Drosophila (15, 16), cultured human cells (17, 18), and mice (19), it is becoming clear that, whereas both h-mtTFB1 and h-mtTFB2 can bind POLRMT and activate transcription in vitro, h-mtTFB2 is probably the primary transcription factor in vivo, whereas h-mtTFB1 is the primary rRNA methyltransferase critical for mitochondrial ribosome biogenesis and translation. However, both proteins have retained both activities (20, 21) and act in concert to promote normal mitochondrial biogenesis, gene expression, and activity (17). In summary, it is now generally accepted that the core machinery needed for mitochondrial transcription initiation is POLRMT, h-mtTFB2, and h-mtTFA, which are all needed together to obtain promoter-specific initiation (22). However, the nature of transcription complexes in vivo remains largely undetermined and the involvement of other factors is clear. For example, the MTERF family of proteins regulates various aspects of transcription (4, 2325) and human MRPL12, in addition to its role in mitochondrial ribosomes, binds directly to POLRMT and activates transcription in mitochondrial lysates (26).

The organizational units of mtDNA in vivo are nucleoids (27), which are protein–DNA complexes that contain 2–10 mtDNA molecules (28) that can be visualized with fluorescence microscopy as puncta in the organelle matrix (29). Mammalian nucleoid composition has been analyzed by several groups (30, 31) and a common component identified is h-mtTFA, which is thought to be a major packaging component in addition to a transcription factor (32). However, in yeast and mammals, nucleoids are also associated with other proteins (3336) and exist as heterogeneous populations (35, 36). At present differential nucleoid structure and function are poorly understood, but it is reported that only specific subsets of nucleoids (or mtDNA molecules) are undergoing transcription and replication at a given point in time (28, 37, 38). Relevant to this study, nucleoids vary significantly in the amount of h-mtTFA associated in vivo (37), which could represent a link between nucleoid structure and the propensity for transcription and replication.

In budding yeast, transcription initiation from mtDNA promoters in vitro requires only Rpo41p and Mtf1p (39, 40), the yeast orthologs of POLRMT and h-mtTFB2, respectively, despite the fact that an ortholog of h-mtTFA (Abf2p) exists. In yeast, Abf2p is involved in mtDNA packaging (35, 41) and oxidative DNA damage resistance (42) and is missing the C-terminal extension found in the human protein required for its transcriptional stimulatory activity (43, 44). This has led to the generalization that the human and yeast basal mitochondrial transcription systems are quite diverged with yeast being a two-component system and human being a three-component system that is dependent on h-mtTFA as an obligate member of the initiation complex (7, 22, 40, 45, 46). In this study, using a recombinant human mitochondrial transcription system, we show that promoter-specific initiation of transcription by POLRMT and h-mtTFB2 can occur in the absence of h-mtTFA in vitro, providing important new insight into the evolution of the human mitochondrial transcription machinery and its regulation.


A Faithful, Fully Recombinant Human Mitochondrial Transcription System with All Proteins Purified from Escherichia coli.

To better understand the regulation of human mitochondrial transcription, we have taken advantage of a recombinant system in which three major components required for initiation from the LSP and HSP1 promoters in vitro (POLRMT, h-mtTFB2, and h-mtTFA), were purified individually from E. coli to near homogeneity (47). In an in vitro reaction using these components, in conjunction with a linear transcription template containing both LSP and HSP1 promoters (Fig. 1A), we were able to generate the predicted full-length (267 nt) run-off product from HSP1 and the full-length (182 nt) run-off product from the LSP (Fig. 1B). We also observed the approximately 120 nt truncated product from the LSP described by Falkenberg and colleagues (48), caused by a strong pause or termination site downstream of conserved sequence block 2 (CSB2, Fig. 1 A and B). When a template missing CSBII was used this termination product was no longer observed and instead only two major products are produced (Fig. 1C). All of these results were obtained using similar protein and template concentrations that Falkenberg et al. (14) used with their recombinant transcription system (where POLRMT and h–mtTFB2 are copurified from insect cells instead of E. coli) and very similar results were obtained with regard to the relative activities of the HSP1 and LSP promoters and the presence of the truncated LSP product. Thus, we are confident that this transcription system is accurately recapitulating the human mitochondrial transcription initiation events involving POLRMT, h-mtTFB2, and h-mtTFA in vitro.

Fig. 1.
A recombinant human mitochondrial transcription system from E.coli recapitulates promoter-specific initiation from linear DNA templates containing both LSP and HSP1. (A) Schematic representation of the D-loop regulatory region of human mtDNA, with salient ...

Initiation of Transcription at HSP1 and LSP in the Absence of h-mtTFA.

It has been clearly established that h-mtTFA significantly activates transcription from the human HSP1 and LSP promoters. However, in budding yeast, promoter-specific mitochondrial transcription initiation in vitro requires only Rpo41p and Mtf1p, the yeast orthologs of POLRMT and h-mtTFB2 (21), leaving open the possibility that this may also be the case in higher organisms. Accordingly, we observed significant transcription activity when only POLRMT and h-mtTFB2 were added to the reaction (Fig. 2A). That is, in contrast to a previous report (14), promoter-specific transcription can occur in vitro at LSP and HSP1 in the complete absence of h-mtTFA. By keeping the concentration of POLRMT and template constant and varying the amount of h-mtTFB2 added to the reaction, we were able to demonstrate that this newly identified h-mtTFA-independent activity is dose-dependent with regard to h-mtTFB2 (Fig. 2B). Furthermore, depending on the precise protein concentrations used, h-mtTFA-independent initiation at HSP1 was equal to or more efficient than at LSP (Fig. 2 A and B). This situation was largely reversed when h-mtTFA was added to the reaction with transcription from LSP becoming more efficient than HSP1 (Fig. 2B, compare lanes 4 and 5). Similar results were observed on longer transcription templates, indicating that the ability of POLRMT/h-mtTFB2 to initiate transcription without h-mtTFA is not an artifact of using shorter transcription templates (Fig. S1). Finally, to address in a different manner that h-mtTFA was indeed responsible for the h-mtTFA-dependent switch in relative promoter utilization from HSP1 to LSP, we added antibody against h-mtTFA to a transcription reaction containing all three components (POLRMT, h-mtTFB2, and h-mtTFA) that preferentially activated the LSP. As predicted, addition of anti-h-mtTFA antibody preferentially inhibited transcription from LSP (Fig. 2C), closely resembling reactions that only contain POLRMT and h-mtTFB2.

Fig. 2.
Promoter-specific initiation of transcription at HSP1 and LSP by POLRMT and h-mtTFB2 in the absence of h-mtTFA. (A) Autoradiogram of transcription products obtained in run-off transcription reactions using template LSP3.1 (see Fig. 1A) and POLRMT ...

Differential Responses of the LSP and HSP1 to Altered Amounts of h-mtTFA.

Given the influence adding h-mtTFA to the POLRMT/h-mtTFB2 reaction had on switching relative HSP1 and LSP promoter utilization in vitro (Fig. 2A), we examined in more detail how different concentrations of h-mtTFA affect transcription from these two promoters. Whereas HSP1-driven transcription equals or exceeds that of LSP in the complete absence of h-mtTFA (as already shown, Fig. 2B), when as little as 4 nM of h-mtTFA is present in the reaction, LSP-driven transcription now prevails (Fig. 3). Furthermore, adding increasing amounts of h-mtTFA to the reaction activated both HSP1 and LSP transcription further. However, the h-mtTFA concentration required to begin to stimulate HSP1 (between 20 and 40 nM) was significantly higher for HSP1 than that for LSP (4 nM or less; Fig. 3). In addition, the maximal amount of stimulation of LSP by h-mtTFA was significantly greater than for HSP1 (e.g., compare LSP at 200 nM and HSP1 at 400 nM). Altogether, these results show that the concentration of h-mtTFA is a critical parameter that determines the relative amount of initiation from the HSP1 and LSP promoters.

Fig. 3.
Differential dose response of HSP1 and LSP transcription to increasing amounts of h-mtTFA. Shown is an autoradiogram of representative transcripts produced in response to increasing concentrations of h-mtTFA (4, 20, 40, 200, 400, 600, and 800 nM ...

POLRMT/h-mtTFB2 Can Initiate Transcription on Isolated HSP1 and LSP Promoters In Vitro, But the Response to h-mtTFA Is Influenced By their Competition for Factors and Interpromoter Communication Through their cis-Arrangement.

Thus far, our analysis involved transcription templates that contained both HSP1 and LSP with natural spacing (i.e., with the normal interpromoter region) and significant additional mtDNA sequences downstream of the promoters. Because h-mtTFA has nonspecific DNA-binding activity, we also assayed h-mtTFA-independent and h-mtTFA-dependent transcription on isolated HSP1 and LSP promoter templates (containing only the initiator sequences and the single documented h-mtTFA binding site) that are less prone to effects of nonspecific h-mtTFA binding. Using these templates in an RNA-primed transcription reaction as described in refs. 47 and 49, we first confirmed that each promoter in isolation was capable of initiating transcription in the absence of h-mtTFA and found that HSP1 is more active than LSP under these conditions (Fig. 4A). However, the promoters responded quite differently to h-mtTFA than we observed previously with the longer, dual-promoter templates in the standard run-off assay (compare to Fig. 3). This prompted us to construct an analogous dual-promoter template that yields similar short run-off products from HSP1 and LSP and assay it under identical conditions (Fig. 4A). When the relative activity of the promoters in isolation are compared to those in the analogous dual-promoter configuration (Fig. 4B) we found that on the dual-promoter template (i) the LSP was stimulated over a wider range of h-mtTFA concentrations and exhibited a greater amplitude response, (ii) more h-mtTFA was needed for maximal stimulation of HSP1, and (iii) both the h-mtTFA-independent and h-mtTFA-dependent activity of HSP1 was greater.

Fig. 4.
Isolated LSP and HSP1 templates support specific h-mtTFA-independent transcription initiation, but promoter responses to h-mtTFA are altered when both promoters are present on the same template. (A) Shown are results from RNA-trinucleotide-primed transcription ...

To address which of the effects observed in the dual-promoter context were due to direct competition between the promoters for transcription components, we analyzed transcription in reactions that contained both promoters in trans as opposed to linked together (Fig. S3). Here, compared to the analogous isolated promoter reactions, we observed a similar right shift in the dose response of HSP1 to h-mtTFA as seen in the dual-promoter context, indicative of promoter competition. This response was not observed when HSP1 was competed with a random, nonspecific DNA competitor (Fig. S4). However, in contrast to the dual-promoter, having the promoters present in trans did not result in enhanced activation of HSP1 and LSP compared to the isolated promoter reactions (Fig. S3), suggesting some form of communication between the promoters in cis is responsible for this phenomenon.


The first major finding of this study is that specific initiation of transcription at the human mitochondrial promoters, HSP1 and LSP, can occur in the absence of h-mtTFA in vitro. As we will discuss, this observation has broad-reaching implications for understanding human mitochondrial transcription from both a mechanistic and evolutionary perspective.

There is extensive documentation that h-mtTFA is required for efficient transcription initiation from the human mitochondrial HSP1 and LSP promoters in vitro (14, 5053) and our results herein certainly confirm this. However, we demonstrate that POLRMT and h-mtTFB2 can carry out specific initiation from HSP1 and LSP in vitro. Recombinant human transcription systems have been developed previously (14, 49, 52) that are capable of assessing the individual contributions of the known core components (POLRMT, h-mtTFB1, h-mtTFB2, and h-mtTFA), however the ability of h-mtTFB2 and POLRMT alone (i.e., in the absence of h-mtTFA) to initiate promoter-specific transcription has not been acknowledged previously. In fact, Gaspari et al. (53) reported that mitochondrial RNA polymerase/h-mtTFB2 complexes cannot initiate normal or abortive transcription from the LSP in vitro and concluded that h-mtTFA is an obligate member of mitochondrial transcription initiation complexes. Our results are inconsistent with this conclusion and instead indicate that the core human transcription machinery is a two-component system in vitro. The reasons for this discrepancy are unclear, but may lie in the fact that HSP1, which is more robust with regard to h-mtTFA-independent activity than LSP under many assay conditions, was not systematically analyzed for h-mtTFA-independent activity and/or the use of epitope-tagged POLRMT and h-mtTFB2 that were copurified from insect cells (as opposed to purified individually from E. coli in an untagged form as was done herein) was confounding.

The h-mtTFA-independent promoter initiation activity of POLRMT and h-mtTFB2 identified implies that the requirements for initiation at the LSP and HSP1 are even more distinct than documented previously (14, 22, 50, 54). This is highlighted in titration experiments with h-mtTFA, in which HSP1 and LSP respond very differently to increasing concentrations of h-mtTFA in the reaction (Figs. 3 and and4).4). That is, relative promoter utilization varies as a function of the amount of h-mtTFA present. These differences are especially intriguing when the distinct roles for LSP and HSP1 transcripts are considered. Transcription from LSP, in addition to providing a single mRNA (ND6) and 8 tRNAs, also produces primers required to initiate mtDNA replication from the heavy-strand origin (6, 7). Conversely, transcription from HSP1 primarily generates the two rRNAs required for mitochondrial ribosome biogenesis and translation. Thus, regulated changes in the steady-state amounts of h-mtTFA could determine the relative commitment of overall mitochondrial transcription to mtDNA replication versus gene expression by differentially activating HSP1 and LSP. Such models have been proposed before (3, 11, 55, 56) and our results provide additional experimental support for this concept. Altogether, our results point to intricate regulation of the HSP1 and LSP promoters by a basal two-component system that is highly sensitive to how much h-mtTFA is bound per template, at least in vitro. Furthermore, the fact that efficiency of transcription from both promoters in vitro is altered when assayed as separate units, as opposed to together on the same template (Fig. 4), suggests there is a physical interaction between the promoters and/or additional cis-acting elements that determine the precise responses to h-mtTFA. Because the major difference between the isolated promoter templates and the dual-promoter template analyzed in Fig. 4 is the presence of the interpromoter region, ours results implicate this region as an important determinant of how h-mtTFA influences transcription from both promoters. This form of regulation appears to work in concert with the LSP competing transcription components away from HSP1, which we show is responsible for the rightward shift in the dose-response of HSP1 to h-mtTFA when the two promoters are present in cis (Fig. 4) or in trans in the same reaction (Fig. S3). It is noteworthy that, even when h-mtTFA is present at levels where LSP activation is high due to its outcompeting HSP1, there remains significant HSP1 transcription that is h-mtTFA-independent in vitro on the dual-promoter template (Fig. 4; 25–200 nM h-mtTFA). This provides proof of principle that an h-mtTFA-independent activity at HSP1 could be relevant in vivo, where h-mtTFA is present.

Mounting evidence indicates that nucleoids are heterogeneous with regard to structure, replication, and transcription (28, 30, 37). For example, it has been postulated that not all nucleoids (or individual molecules within nucleoids) are undergoing active transcription and/or replication. Furthermore, when nucleoids are assayed for the presence of mtTFA by microscopy, there is often a mixture of mtTFA-positive and mtTFA-negative puncta, indicating that there is internucleoid variability with regard to mtTFA binding (37). Though at this point we must remain cautious in extrapolating the in vitro results obtained herein to the situation in vivo, it is in this context we propose that the ability of POLRMT/h-mtTFB2 complexes to initiate transcription in the absence of h-mtTFA and the differential response of the LSP and HSP1 promoters to h-mtTFA binding may be relevant in vivo. Specifically, in nucleoids that have little or no h-mtTFA associated, the ability of POLRMT/h-mtTFB2 to initiate transcription independently of h-mtTFA could be important to allow them to remain transcriptionally active. In addition, the preferential activation of POLRMT/h-mtTFB2 at LSP by h-mtTFA could allow nucleoids that have less h-mtTFA to preferentially undergo transcription-primed replication. How h-mtTFA nucleoid binding is regulated and its potential role in differentially controlling mtDNA transcription and replication remain important outstanding questions. In this regard, it is noteworthy that there are conflicting reports on relative abundance of h-mtTFA and mtDNA in vivo, ranging from 35–50 to approximately 3,000 h-mtTFA molecules per mtDNA molecule (18, 57, 58). Based on our results, these extremes could have very different consequences on relative promoter utilization and replication-priming capacity. More attention is clearly warranted in this area and determining how mtTFA is distributed between nucleoids, or between mtDNA molecules within nucleoids, seems particularly important to address (as opposed to simply determining the overall mtTFA/mtDNA ratios).

The ability of POLRMT/h-mtTFB2 complexes to alone initiate transcription in vitro makes sense in terms of the evolution of mitochondrial transcription systems. Specifically, it has been known for many years that, despite the presence of the h-mtTFA ortholog in budding yeast, Abf2p, specific initiation of transcription from a yeast mtDNA promoter in vitro requires only Rpo41p and Mtf1p (39, 40), the yeast orthologs of POLRMT and h-mtTFB2, respectively. Even more like the ancestral T7 RNA polymerase, plant POLRMT homologues exhibit in vitro transcription activity without any other protein factors (59). The independence from mitochondrial transcription factors in plants is consistent with a previous study that failed to identify an mtTFB homologue in plants (60). Thus, at a very fundamental level, the yeast and human systems are indeed more alike than previously appreciated. Based on our results, we propose that in a common ancestor of Fungi and Metazoa the ancestral homologue of h-mtTFB2 (i.e., the endosymbiont-derived KsgA methyltransferase) acquired its role as a transcription factor for the mitochondrial RNA polymerase. Meanwhile, the ancestral homologue of h-mtTFA/Abf2p was initially important for mtDNA packaging, but at some point in metazoan evolution, mtTFA acquired the ability to stimulate transcription through the acquisition of a C-terminal tail (43) and coevolution of specific binding sites in mtDNA adjacent to promoters. This also likely allowed h-mtTFA to acquire a more exquisite ability to regulate the multifunctional LSP, which has a role in priming mtDNA replication as well as transcription (68).

Methods and Materials

Recombinant Proteins.

Purified recombinant POLRMT (aa 42–1260), h-mtTFA (aa 42–246), and h-mtTFB2 (aa 31–396) are available from Enzymax, LLC. Each of these proteins is expressed in E. coli without its predicted mitochondrial-targeting sequence and purified by conventional chromatography techniques without the use of epitope tags (47).

Mitochondrial Run-Off Transcription Assays.

In Figs 13, the linear DNA templates for simultaneously analyzing transcription from HSP1 and LSP (Fig. 1A) were PCR products corresponding to human mtDNA sequence 242–825 (LSP3) or 317–825 (LSP3.1) cloned into the pGEMT-EZ (Promega, Inc.) (26). For use in the transcription reactions, the mtDNA template was liberated from the vector by digestion with EcoR1 (LSP3) or NotI (LSP3.1), gel-purified, and quantified by OD260. Transcription reaction were performed in a total volume of 25 μL containing 10 mM Tris-Cl (pH 8.0), 10–20 mM MgCl2, 100 μM DTT, 100 μg/mL BSA, 400 μM ATP, 150 μM CTP, 150 μM GTP, 10 μM UTP, 0.2 μM [α-32P]UTP (3,000 Ci/mmol; Perkin Elmer), template DNA (3.4 nM), and 4 units of RNAseOut (InVitrogen, Inc). Concentrations of the protein components, POLRMT, h-mtTFA and h-mtTFB2, are listed in the corresponding figure legends describing each experiment. Reactions were carried out at 32 °C for 30 min and stopped by addition of 22.5 μg proteinase K in 100 μL of Stop Buffer (10 mM Tris-Cl, pH8.0, 0.2 M NaCl, 0.5% SDS, 0.1 μg/μL linear polyacrylamide) followed by incubation at 42 °C for 1 h. Radiolabelled transcription products were precipitated in ethanol, dried, and suspended in 25 μL of gel loading buffer (98% formamide, 10 mM EDTA, pH 8.0, 0.025% xylene cyanol, 0.025% bromophenol blue). Samples were then separated on 5% polyacrylamide/7 M urea gels. The 10 bp DNA ladder (InVitrogen™) used to estimate transcript size was end-labeled according to the manufacturer’s protocol. Protein A-purified h-mtTFA antibody was added to the reaction where indicated. Gels were exposed to X-ray film or quantified directly using a phosphoimager and ImageQuant 5.2 Software. Multiple exposures were analyzed to ensure linearity. The results presented in Fig. 3 were quantified by phosphoimager analysis and the gels used are shown in Fig. S2.

The RNA trinucleotide-primed transcription reactions in Fig. 4 were performed as described (47). The dual-promoter template (corresponding to mtDNA sequences 373–605) was constructed by bridge-ligating three individual oligonucleotides for each strand and then annealing each 233 nt, gel-purified ligation product to form the corresponding double-stranded 233 bp template used in the transcription reactions.

Supplementary Material

Supporting Information:


The authors thank Dr. David A. Clayton for providing h-mtTFA antibodies, and Megan Bestwick and Yulia Surovtseva for their help and feedback on the project. This work was supported by National Institutes of Health National Heart, Lung, and Blood Institute Grant HL-059655 (G.S.S.) and by the United Mitochondrial Disease Foundation (T.E.S.). The mitochondrial transcription studies in the lab of C.E.C. are supported by the Berg Endowment of the Eberly College of Science.

Note Added in Proof.

Following the acceptance of our manuscript, we became aware of the study by Temiakov and coworkers (61) in which they also show substantial transcription initiation from both HSP1 and LSP in the presence of only POLRMT and h-mtTFB2 in vitro (i.e., in the absence of h-mtTFA). While these authors appear to discount this activity as relevant in vitro or in vivo, we respectfully disagree and, instead, view their results as confirmatory of the two-component core human mitochondrial transcription complex we have elucidated herein.


Conflict of interest statement: C.E.C. has a relationship with Enzymax, the company that markets the new human mitochondrial transcription system.

This article is a PNAS Direct Submission. D.A.C. is a guest editor invited by the Editorial Board.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.0910581107/-/DCSupplemental.


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