Transcriptional requirements of the distal heavy-strand promoter of mtDNA
Edited by Douglas C. Wallace, Center for Mitochondrial and Epigenomic Medicine, Children's Hospital of Philadelphia, Philadelphia, PA, and approved March 6, 2012 (received for review November 12, 2011)
Abstract
The heavy strand of mtDNA contains two promoters with nonoverlapping functions. The role of the minor heavy-strand promoter (HSP2) is controversial, because the promoter has been difficult to activate in an in vitro system. We have isolated HSP2 by excluding its interaction with the more powerful HSP1 promoter, and we find that it is transcribed efficiently by recombinant mtRNA polymerase and mitochondrial transcription factor B2. The mitochondrial transcription factor A is not required for initiation, but it has the ability to alternatively activate and repress the HSP2 transcriptional unit depending on the ratio between mitochondrial transcription factor A and other transcription factors. The positioning of transcriptional initiation agrees with our current understanding of HSP2 activity in vivo. Serial deletion of HSP2 shows that only proximal sequences are required. Several mutations, including the disruption of a polycytosine track upstream of the HSP2 initiation site, influence transcriptional activity. Transcription from HSP2 is also observed when HeLa cell mitochondrial extract is used as the source of mitochondrial polymerase, and this transcription is maintained when HSP2 is provided in proper spacing and context to the HSP1 promoter. Studies of the linked heavy-strand promoters show that they are differentially regulated by ATP dosage. We conclude that HSP2 is transcribed and has features that allow it to regulate mitochondrial mRNA synthesis.
Acknowledgments
Craig Cameron, Fernanda Lodeiro, and Brett Kaufman read drafts of the manuscript. Eleonara Lamantea provided technical assistance. Support for this work was provided by National Institutes of Health Grant K08-HD58022 (to N.S.) as well as the Children's Hospital of Philadelphia Pediatric Development Fund.
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References
1
S Anderson, et al., Sequence and organization of the human mitochondrial genome. Nature 290, 457–465 (1981).
2
DD Chang, DA Clayton, Priming of human mitochondrial DNA replication occurs at the light-strand promoter. Proc Natl Acad Sci USA 82, 351–355 (1985).
3
R Gelfand, G Attardi, Synthesis and turnover of mitochondrial ribonucleic acid in HeLa cells: The mature ribosomal and messenger ribonucleic acid species are metabolically unstable. Mol Cell Biol 1, 497–511 (1981).
4
J Montoya, GL Gaines, G Attardi, The pattern of transcription of the human mitochondrial rRNA genes reveals two overlapping transcription units. Cell 34, 151–159 (1983).
5
J Montoya, T Christianson, D Levens, M Rabinowitz, G Attardi, Identification of initiation sites for heavy-strand and light-strand transcription in human mitochondrial DNA. Proc Natl Acad Sci USA 79, 7195–7199 (1982).
6
BK Yoza, DF Bogenhagen, Identification and in vitro capping of a primary transcript of human mitochondrial DNA. J Biol Chem 259, 3909–3915 (1984).
7
M Martin, J Cho, AJ Cesare, JD Griffith, G Attardi, Termination factor-mediated DNA loop between termination and initiation sites drives mitochondrial rRNA synthesis. Cell 123, 1227–1240 (2005).
8
D Litonin, et al., Human mitochondrial transcription revisited: Only TFAM and TFB2M are required for transcription of the mitochondrial genes in vitro. J Biol Chem 285, 18129–18133 (2010).
9
TE Shutt, MF Lodeiro, J Cotney, CE Cameron, GS Shadel, Core human mitochondrial transcription apparatus is a regulated two-component system in vitro. Proc Natl Acad Sci USA 107, 12133–12138 (2010).
10
JE Hixson, DA Clayton, Initiation of transcription from each of the two human mitochondrial promoters requires unique nucleotides at the transcriptional start sites. Proc Natl Acad Sci USA 82, 2660–2664 (1985).
11
L Valente, et al., Identification of novel mutations in five patients with mitochondrial encephalomyopathy. Biochim Biophys Acta 1787, 491–501 (2009).
12
JA Enríquez, P Fernández-Silva, A Pérez-Martos, MJ López-Pérez, J Montoya, The synthesis of mRNA in isolated mitochondria can be maintained for several hours and is inhibited by high levels of ATP. Eur J Biochem 237, 601–610 (1996).
13
N Sondheimer, JK Fang, E Polyak, MJ Falk, NG Avadhani, Leucine-rich pentatricopeptide-repeat containing protein regulates mitochondrial transcription. Biochemistry 49, 7467–7473 (2010).
14
L Liu, et al., LRP130 protein remodels mitochondria and stimulates fatty acid oxidation. J Biol Chem 286, 41253–41264 (2011).
15
F Xu, C Morin, G Mitchell, C Ackerley, BH Robinson, The role of the LRPPRC (leucine-rich pentatricopeptide repeat cassette) gene in cytochrome oxidase assembly: Mutation causes lowered levels of COX (cytochrome c oxidase) I and COX III mRNA. Biochem J 382, 331–336 (2004).
16
MF Lodeiro, et al., Identification of multiple rate-limiting steps during the human mitochondrial transcription cycle in vitro. J Biol Chem 285, 16387–16402 (2010).
17
M Gaspari, M Falkenberg, NG Larsson, CM Gustafsson, The mitochondrial RNA polymerase contributes critically to promoter specificity in mammalian cells. EMBO J 23, 4606–4614 (2004).
18
BA Kaufman, et al., The mitochondrial transcription factor TFAM coordinates the assembly of multiple DNA molecules into nucleoid-like structures. Mol Biol Cell 18, 3225–3236 (2007).
19
TI Alam, et al., Human mitochondrial DNA is packaged with TFAM. Nucleic Acids Res 31, 1640–1645 (2003).
20
RP Fisher, T Lisowsky, MA Parisi, DA Clayton, DNA wrapping and bending by a mitochondrial high mobility group-like transcriptional activator protein. J Biol Chem 267, 3358–3367 (1992).
21
CS Malarkey, M Bestwick, JE Kuhlwilm, GS Shadel, ME Churchill, Transcriptional activation by mitochondrial transcription factor A involves preferential distortion of promoter DNA. Nucleic Acids Res 40, 614–624 (2012).
22
HB Ngo, JT Kaiser, DC Chan, The mitochondrial transcription and packaging factor Tfam imposes a U-turn on mitochondrial DNA. Nat Struct Mol Biol 18, 1290–1296 (2011).
23
RP Fisher, DA Clayton, Purification and characterization of human mitochondrial transcription factor 1. Mol Cell Biol 8, 3496–3509 (1988).
24
TE Shutt, M Bestwick, GS Shadel, The core human mitochondrial transcription initiation complex: It only takes two to tango. Transcription 2, 55–59 (2011).
25
Lodeiro, et al. Proc Natl Acad Sci USA 109, 6513–6518 (2012).
26
EA Amiott, JA Jaehning, Mitochondrial transcription is regulated via an ATP “sensing” mechanism that couples RNA abundance to respiration. Mol Cell 22, 329–338 (2006).
27
E Metelkin, O Demin, Z Kovács, C Chinopoulos, Modeling of ATP-ADP steady-state exchange rate mediated by the adenine nucleotide translocase in isolated mitochondria. FEBS J 276, 6942–6955 (2009).
28
C Glatz, K D'Aco, S Smith, N Sondheimer, Mutation in the mitochondrial tRNA(Val) causes mitochondrial encephalopathy, lactic acidosis and stroke-like episodes. Mitochondrion 11, 615–619 (2011).
29
JM Greene, K Struhl, S1 analysis of messenger RNA using single-stranded DNA probes. Curr Protoc Mol Biol, 2001).
30
RL Dorit, O Ohara, cDNA amplification using one-sided (anchored) PCR. Curr Protoc Mol Biol, 2001).
Information & Authors
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Cover image: Pictured are HeLa cells in various stages of the cell division cycle, displaying the nucleus (stained blue) and a protein called “transferrin receptor” (stained white when localized on the cell surface and red when internalized into cells through a process called “clathrin-mediated endocytosis”). Andrew B. Fielding et al. report that nondividing cells in a stage of the cell cycle called interphase internalize the transferrin receptor; whereas dividing cells temporarily halt endocytosis, as seen in the lone, rounded cell at the center whose receptor molecules are largely clustered at the surface. The findings suggest that endocytosis does not continue unabated during cell division. See the article by Fielding et al. on pages 6572–6577. Image courtesy of Andrew B. Fielding.
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Submission history
Published online: March 27, 2012
Published in issue: April 24, 2012
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Acknowledgments
Craig Cameron, Fernanda Lodeiro, and Brett Kaufman read drafts of the manuscript. Eleonara Lamantea provided technical assistance. Support for this work was provided by National Institutes of Health Grant K08-HD58022 (to N.S.) as well as the Children's Hospital of Philadelphia Pediatric Development Fund.
Notes
*This Direct Submission article had a prearranged editor.
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Competing Interests
The authors declare no conflict of interest.
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