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Core Human Mitochondrial Transcription Apparatus Is a Regulated Two-Component System in Vitro

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Core Human Mitochondrial Transcription Apparatus Is a Regulated Two-Component System in Vitro Core human mitochondrial transcription apparatus is a regulated two-component system in vitro Timothy E. Shutta, Maria F. Lodeirob, Justin Cotneya, Craig E. Cameronb, and Gerald S. Shadela,c,1 aDepartment of Pathology, Yale University School of Medicine, 310 Cedar Street, P.O. Box 208023, New Haven, CT 06520-8023; bDepartment of Biochemistry and Molecular Biology, Pennsylvania State University, 201 Althouse Laboratory, University Park, PA 16802; and cDepartment of Genetics, Yale University School of Medicine, 333 Cedar Street, P.O. Box 208005, New Haven, CT 06520-8005 Edited by David A. Clayton, Howard Hughes Medical Institute, Ashburn, VA, and accepted by the Editorial Board May 25, 2010 (received for review September 15, 2009) The core human mitochondrial transcription apparatus is currently ribosomal proteins to generate mitochondrial ribosomes. Finally, regarded as an obligate three-component system comprising the transcripts from the LSP are also used as primers for mtDNA bacteriophage T7-related mitochondrial RNA polymerase, the rRNA replication (5, 6), thus LSP transcription serves a dual role in methyltransferase-related transcription factor, h-mtTFB2, and the gene expression and mtDNA maintenance (7, 8). high mobility group box transcription/DNA-packaging factor, Much effort has been devoted to identifying the core machin- h-mtTFA/TFAM. Using a faithful recombinant human mitochondrial ery needed for mitochondrial transcription in humans as a critical transcription system from Escherichia coli, we demonstrate that step toward understanding the mechanism of human mitochon- specific initiation from the mtDNA promoters, LSP and HSP1, only drial gene expression and replication in vivo and its role in human requires mitochondrial RNA polymerase and h-mtTFB2 in vitro. disease. The human mitochondrial RNA polymerase (POLRMT) When h-mtTFA is added to these basal components, LSP exhibits is a single-subunit enzyme related to the T7 family of bacterioph- a much lower threshold for activation and a larger amplitude re- age RNA polymerases (9, 10). However, unlike T7 RNA polymer- sponse than HSP1. In addition, when LSP and HSP1 are together ase, which does not require any transcription factors, efficient on the same transcription template, h-mtTFA-independent tran- promoter-specific initiation by human POLRMT in vitro requires scription from HSP1 and h-mtTFA-dependent transcription from the high mobility group box transcription factor h-mtTFA/TFAM both promoters is enhanced and a higher concentration of h-mtTFA (referred to as h-mtTFA from this point forward) and one of two is required to stimulate HSP1. Promoter competition experiments rRNA methyltransferase-related transcription factors, h-mtTFB1 – Drosophila revealed that, in addition to LSP competing transcription compo- and h-mtTFB2 (11 14). Based on work in (15, 16), nents away from HSP1, additional cis-acting signals are involved cultured human cells (17, 18), and mice (19), it is becoming clear in these aspects of promoter regulation. Based on these results, that, whereas both h-mtTFB1 and h-mtTFB2 can bind POLRMT we speculate that the human mitochondrial transcription system and activate transcription in vitro, h-mtTFB2 is probably the pri- may have evolved to differentially regulate transcription initiation mary transcription factor in vivo, whereas h-mtTFB1 is the primary and transcription-primed mtDNA replication in response to the rRNA methyltransferase critical for mitochondrial ribosome amount of h-mtTFA associated with nucleoids, which could begin biogenesis and translation. However, both proteins have retained to explain the heterogeneity of nucleoid structure and activity in both activities (20, 21) and act in concert to promote normal vivo. Furthermore, this study sheds new light on the evolution of mitochondrial biogenesis, gene expression, and activity (17). In mitochondrial transcription components by showing that the hu- summary, it is now generally accepted that the core machinery man system is a regulated two-component system in vitro, and needed for mitochondrial transcription initiation is POLRMT, thus more akin to that of budding yeast than thought previously. h-mtTFB2, and h-mtTFA, which are all needed together to obtain promoter-specific initiation (22). However, the nature of tran- scription complexes in vivo remains largely undetermined h-mtTFA/TFAM ∣ mtDNA ∣ nucleoid ∣ POLRMT ∣ h-mtTFB2/TFB2M BIOCHEMISTRY and the involvement of other factors is clear. For example, the MTERF family of proteins regulates various aspects of transcrip- uman mtDNA encodes 37 essential genes required for oxi- tion (4, 23–25) and human MRPL12, in addition to its role in Hdative phosphorylation, mutations in which cause maternally mitochondrial ribosomes, binds directly to POLRMTand activates inherited diseases and are also thought to contribute to other transcription in mitochondrial lysates (26). more common disorders, aging, and age-related pathology The organizational units of mtDNA in vivo are nucleoids (27), (1, 2). Expression and replication of mtDNA is completely depen- which are protein–DNA complexes that contain 2–10 mtDNA dent on the nucleus, because all of the regulatory factors required molecules (28) that can be visualized with fluorescence microscopy are encoded by nuclear genes and imported into the organelle. as puncta in the organelle matrix (29). Mammalian nucleoid com- For example, mitochondrial gene expression alone involves position has been analyzed by several groups (30, 31) and a >100 nuclear genes, including a dedicated mitochondrial RNA common component identified is h-mtTFA, which is thought to polymerase and associated transcription factors, RNA processing be a major packaging component in addition to a transcription machinery, and the large cadre of proteins involved in mitochon- factor (32). However, in yeast and mammals, nucleoids are also drial translation. associated with other proteins (33–36) and exist as heterogeneous 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 promo- Author contributions: T.E.S., M.F.L., J.C., C.E.C., and G.S.S. designed research; T.E.S., M.F.L., and J.C. performed research; C.E.C. contributed new reagents/analytic tools; T.E.S., M.F.L., ters for transcription. Transcripts corresponding to both strands J.C., and C.E.C. analyzed data; and T.E.S., M.F.L., and G.S.S. wrote the paper. of mtDNA are initiated from three promoters designated as the Conflict of interest statement: C.E.C. has a relationship with Enzymax, the company that light-strand promoter (LSP) and the heavy-strand promoters 1 markets the new human mitochondrial transcription system. and 2 (HSP1 and HSP2) (3). Transcription from the LSP and This article is a PNAS Direct Submission. D.A.C. is a guest editor invited by the HSP2 result in long polycistronic products, whereas transcription Editorial Board. from HSP1 produces primarily a truncated transcript encoding 1To whom correspondence should be addressed. E-mail: [email protected]. only the two rRNA species (12S and 16S) and two tRNAs (4). This article contains supporting information online at www.pnas.org/lookup/suppl/ The two rRNAs assemble with nucleus-encoded mitochondrial doi:10.1073/pnas.0910581107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0910581107 PNAS ∣ July 6, 2010 ∣ vol. 107 ∣ no. 27 ∣ 12133–12138 Downloaded by guest on September 25, 2021 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 under- going 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 promo- ters 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 stimu- latory 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 mi- tochondrial 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 in- sight into the evolution of the human mitochondrial transcription Fig. 1. A recombinant human mitochondrial transcription system from machinery and its regulation. E.coli recapitulates promoter-specific initiation from linear DNA templates containing both LSP and HSP1. (A) Schematic representation of the D-loop Results regulatory region of human mtDNA, with salient regulatory elements A Faithful, Fully Recombinant Human Mitochondrial Transcription Sys- indicated, including the hallmark D-loop strand (thick black bent arrow). tem with All Proteins Purified from Escherichia coli. To better under- The LSP, HSP1, and HSP2 are depicted as thin bent arrows. Known binding stand the regulation of human mitochondrial transcription, we sites for h-mtTFA at LSP and HSP1 are indicated as black and gray boxes, have taken advantage of a recombinant system in which three ma- respectively, and conserved sequence blocks (CSB) 1, 2, and 3 are depicted as white boxes. The solid lines below the D-loop region diagram denote jor components required for initiation from the LSP and HSP1 the linear mtDNA templates, LSP3 and LSP3.1, used in the run-off transcrip- promoters in vitro (POLRMT, h-mtTFB2, and h-mtTFA), were tion reactions in B and C, respectively.
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