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Identification Of Multiple Rate-limiting Steps During The Human Mitochondrial Transcription Cycle In Vitro*

M. F. Lodeiro, A. Uchida, J. Arnold, Shelley L Reynolds, I. Moustafa, C. Cameron
Published 2010 · Biology, Medicine

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We have reconstituted human mitochondrial transcription in vitro on DNA oligonucleotide templates representing the light strand and heavy strand-1 promoters using protein components (RNA polymerase and transcription factors A and B2) isolated from Escherichia coli. We show that 1 eq of each transcription factor and polymerase relative to the promoter is required to assemble a functional initiation complex. The light strand promoter is at least 2-fold more efficient than the heavy strand-1 promoter, but this difference cannot be explained solely by the differences in the interaction of the transcription machinery with the different promoters. In both cases, the rate-limiting step for production of the first phosphodiester bond is open complex formation. Open complex formation requires both transcription factors; however, steps immediately thereafter only require transcription factor B2. The concentration of nucleotide required for production of the first dinucleotide product is substantially higher than that required for subsequent cycles of nucleotide addition. In vitro, promoter-specific differences in post-initiation control of transcription exist, as well as a second rate-limiting step that controls conversion of the transcription initiation complex into a transcription elongation complex. Rate-limiting steps of the biochemical pathways are often those that are targeted for regulation. Like the more complex multisubunit transcription systems, multiple steps may exist for control of transcription in human mitochondria. The tools and mechanistic framework presented here will facilitate not only the discovery of mechanisms regulating human mitochondrial transcription but also interrogation of the structure, function, and mechanism of the complexes that are regulated during human mitochondrial transcription.
This paper references
10.1146/ANNUREV.BIOCHEM.76.060305.152028
DNA replication and transcription in mammalian mitochondria.
M. Falkenberg (2007)
10.1042/BST0341062
The regulatory roles and mechanism of transcriptional pausing.
R. Landick (2006)
10.1002/9780470725207.CH15
Mitochondrial mutations: genotype to phenotype.
E. Schon (2007)
10.1006/BBRC.1994.1603
Low levels of mitochondrial transcription factor A in mitochondrial DNA depletion.
N. Larsson (1994)
10.1074/jbc.M801342200
Mitochondrial Transcription Factor B2 Is Essential for Metabolic Function in Drosophila melanogaster Development*
C. Adán (2008)
Markedly different ATP requirements for rRNA synthesis and mtDNA light strand transcription versus mRNA synthesis in isolated human mitochondria.
G. Gaines (1987)
10.1016/j.molcel.2008.03.001
Poised polymerases: on your mark...get set...go!
D. Price (2008)
10.1006/METH.1997.0472
Analysis of open complex formation during RNA polymerase II transcription initiation using heteroduplex templates and potassium permanganate probing.
F. Holstege (1997)
10.1093/nar/gkp157
Structural analysis and DNA binding of the HMG domains of the human mitochondrial transcription factor A
T. A. Gangelhoff (2009)
10.1074/jbc.271.48.30451
Equilibrium and Stopped-flow Kinetic Studies of Interaction between T7 RNA Polymerase and Its Promoters Measured by Protein and 2-Aminopurine Fluorescence Changes*
Y. Jia (1996)
10.1146/ANNUREV.MED.59.053006.104646
Inherited mitochondrial diseases of DNA replication.
W. Copeland (2008)
10.1016/S0022-2836(02)01034-3
The energetics of consensus promoter opening by T7 RNA polymerase.
R. P. Bandwar (2002)
10.1073/pnas.0900407106
Mechanism of sequence-specific pausing of bacterial RNA polymerase
M. Kireeva (2009)
10.2144/99273BM11
Single-nucleotide resolution of RNA strands in the presence of their RNA complements.
J. Arnold (1999)
10.1113/eph8802514
Replication and Transcription of Mammalian Mitochondrial Dna
P. Fernández-Silva (2003)
10.1074/jbc.M009901200
Nam1p, a Protein Involved in RNA Processing and Translation, Is Coupled to Transcription through an Interaction with Yeast Mitochondrial RNA Polymerase*
M. Rodeheffer (2001)
10.1016/J.MOLCEL.2006.03.031
Mitochondrial transcription is regulated via an ATP "sensing" mechanism that couples RNA abundance to respiration.
E. Amiott (2006)
10.1016/j.cell.2009.10.031
TFB2 Is a Transient Component of the Catalytic Site of the Human Mitochondrial RNA Polymerase
M. Sologub (2009)
10.1074/jbc.M208405200
Binding of the Priming Nucleotide in the Initiation of Transcription by T7 RNA Polymerase*
I. Kuzmine (2003)
10.1128/MCB.8.8.3496
Purification and characterization of human mitochondrial transcription factor 1.
R. P. Fisher (1988)
10.1016/j.bbabio.2008.10.007
DNA polymerase gamma and mitochondrial disease: understanding the consequence of POLG mutations.
S. Chan (2009)
10.1007/s00412-008-0182-4
Promoter proximal pausing on genes in metazoans
D. Gilmour (2008)
10.1021/BI0158472
Kinetic and thermodynamic basis of promoter strength: multiple steps of transcription initiation by T7 RNA polymerase are modulated by the promoter sequence.
R. P. Bandwar (2002)
10.1016/S0968-0004(01)01801-1
HMG1 and 2, and related 'architectural' DNA-binding proteins.
J. Thomas (2001)
10.1074/jbc.M807880200
Fluorescence Mapping of the Open Complex of Yeast Mitochondrial RNA Polymerase*
Guo-Qing Tang (2009)
10.1093/HUMREP/15.1.11
Urinary oestrogen patterns in long follicular phases.
S. Harlow (2000)
10.1016/j.cmet.2009.03.001
Methylation of 12S rRNA is necessary for in vivo stability of the small subunit of the mammalian mitochondrial ribosome.
Metodi D Metodiev (2009)
10.1074/jbc.M500569200
Drosophila Mitochondrial Transcription Factor B1 Modulates Mitochondrial Translation but Not Transcription or DNA Copy Number in Schneider Cells*
Y. Matsushima (2005)
10.1146/ANNUREV.GENET.39.110304.095751
A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine.
D. Wallace (2005)
10.1038/ng909
Mitochondrial transcription factors B1 and B2 activate transcription of human mtDNA
M. Falkenberg (2002)
10.1093/HMG/DDH109
Mitochondrial transcription factor A regulates mtDNA copy number in mammals.
Mats I Ekstrand (2004)
10.1126/SCIENCE.1059495
Structural Basis of Transcription: An RNA Polymerase II Elongation Complex at 3.3 Å Resolution
A. Gnatt (2001)
10.1006/JMBI.1993.1086
Hierarchy of base-pair preference in the binding domain of the bacteriophage T7 promoter.
G. Diaz (1993)
10.1038/sj.emboj.7600465
The mitochondrial RNA polymerase contributes critically to promoter specificity in mammalian cells
M. Gaspari (2004)
10.1126/science.1131399
Initial Transcription by RNA Polymerase Proceeds Through a DNA-Scrunching Mechanism
A. Kapanidis (2006)
10.1146/ANNUREV.BIOCHEM.76.081205.150955
Why do we still have a maternally inherited mitochondrial DNA? Insights from evolutionary medicine.
D. Wallace (2007)
10.1073/PNAS.0405779102
The effects of upstream DNA on open complex formation by Escherichia coli RNA polymerase.
C. A. Davis (2005)
10.1074/jbc.270.41.24392
Abortive Cycling and the Release of Polymerase for Elongation at the σ54-dependent glnAp2 Promoter (*)
Y. Tintut (1995)
10.1146/annurev.neuro.30.051606.094302
Mitochondrial disorders in the nervous system.
S. Dimauro (2008)
10.1126/science.1169237
Direct Detection of Abortive RNA Transcripts in Vivo
Seth R. Goldman (2009)
10.1128/MCB.23.16.5816-5824.2003
Human Mitochondrial Transcription Factor B1 Interacts with the C-Terminal Activation Region of h-mtTFA and Stimulates Transcription Independently of Its RNA Methyltransferase Activity
Vicki McCulloch (2003)
10.1021/BI982689E
Studies of contacts between T7 RNA polymerase and its promoter reveal features in common with multisubunit RNA polymerases.
C. Place (1999)
10.1074/jbc.M700461200
Human Mitochondrial Ribosomal Protein MRPL12 Interacts Directly with Mitochondrial RNA Polymerase to Modulate Mitochondrial Gene Expression*
Zhibo Wang (2007)
10.1101/gad.1745409
Allosteric control of Escherichia coli rRNA promoter complexes by DksA.
S. Rutherford (2009)
10.1074/jbc.M608638200
Sensitivity of the Yeast Mitochondrial RNA Polymerase to +1 and +2 Initiating Nucleotides*
E. Amiott (2006)
10.1038/nature08449
Defining mechanisms that regulate RNA polymerase II transcription in vivo
Nicholas J. Fuda (2009)
10.1128/MCB.22.4.1116-1125.2002
A Human Mitochondrial Transcription Factor Is Related to RNA Adenine Methyltransferases and Binds S-Adenosylmethionine
Vicki McCulloch (2002)
10.4161/cc.8.16.9305
Divergent transcription: A new feature of active promoters
Amy C. Seila (2009)
10.1074/jbc.274.5.2706
Poliovirus RNA-dependent RNA Polymerase (3Dpol) Is Sufficient for Template Switchingin Vitro *
J. Arnold (1999)
10.1146/annurev.biochem.76.052705.164655
RNA polymerase active center: the molecular engine of transcription.
E. Nudler (2009)
10.1126/SCIENCE.1280856
Functional transcription elongation complexes from synthetic RNA-DNA bubble duplexes.
S. Daube (1992)
A transcription factor required for promoter recognition by human mitochondrial RNA polymerase. Accurate initiation at the heavy- and light-strand promoters dissected and reconstituted in vitro.
R. P. Fisher (1985)
10.1021/BI00433A002
T7 RNA polymerase does not interact with the 5'-phosphate of the initiating nucleotide.
C. Martin (1989)
10.1021/BI00411A012
Processivity in early stages of transcription by T7 RNA polymerase.
C. Martin (1988)
10.1021/BI00384A006
Kinetic analysis of T7 RNA polymerase-promoter interactions with small synthetic promoters.
C. Martin (1987)
10.1074/jbc.M900718200
A Promoter Recognition Mechanism Common to Yeast Mitochondrial and Phage T7 RNA Polymerases*
D. Nayak (2009)
R.,Witherell, G.W., andUhlenbeck,O.C
J. F. Milligan (1987)
10.1016/J.TIBS.2007.01.003
Mitochondrial transcription and its regulation in mammalian cells.
J. Asin-Cayuela (2007)
10.1016/j.cell.2005.09.040
Termination Factor-Mediated DNA Loop between Termination and Initiation Sites Drives Mitochondrial rRNA Synthesis
M. Martín (2005)
10.1006/JMBI.1997.1358
Role of open complex instability in kinetic promoter selection by bacteriophage T7 RNA polymerase.
J. Villemain (1997)
10.1016/0092-8674(87)90220-0
Promoter selection in human mitochondria involves binding of a transcription factor to orientation-independent upstream regulatory elements
R. P. Fisher (1987)
10.1146/ANNUREV.GE.29.120195.001055
Molecular genetic aspects of human mitochondrial disorders.
N. Larsson (1995)
10.1093/NAR/15.21.8783
Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates.
J. Milligan (1987)
10.1016/0167-4781(87)90029-7
Roles for a promoter and RNA processing in the synthesis of mitochondrial displacement-loop strands.
D. D. Chang (1987)
10.1074/jbc.M604023200
Mechanism of Instability in Abortive Cycling by T7 RNA Polymerase*
Peng Gong (2006)
10.1126/SCIENCE.2035027
Similarity of human mitochondrial transcription factor 1 to high mobility group proteins.
M. Parisi (1991)
10.1016/S0959-440X(02)00005-2
Bacterial RNA polymerases: the wholo story.
K. Murakami (2003)
10.1074/jbc.M602429200
Conserved Sequence Box II Directs Transcription Termination and Primer Formation in Mitochondria*
X. H. Pham (2006)
10.1006/JMBI.1993.1458
Termination and slippage by bacteriophage T7 RNA polymerase.
L. Macdonald (1993)
10.1021/BI026954E
In vitro studies of transcript initiation by Escherichia coli RNA polymerase. 1. RNA chain initiation, abortive initiation, and promoter escape at three bacteriophage promoters.
L. Hsu (2003)
10.1074/JBC.M401643200
Drosophila Mitochondrial Transcription Factor B2 Regulates Mitochondrial DNA Copy Number and Transcription in Schneider Cells*
Y. Matsushima (2004)
10.1006/JMBI.1995.9889
Addition of a 29 residue carboxyl-terminal tail converts a simple HMG box-containing protein into a transcriptional activator.
D. J. Dairaghi (1995)
10.1016/j.gde.2007.12.008
The transition from transcriptional initiation to elongation.
J. Wade (2008)
10.1016/J.MOLCEL.2006.11.024
Initiation and beyond: multiple functions of the human mitochondrial transcription machinery.
Nicholas D Bonawitz (2006)
10.1016/J.TIG.2004.08.005
Coupling the mitochondrial transcription machinery to human disease.
G. Shadel (2004)
10.1146/ANNUREV.BIOCHEM.66.1.117
Basic mechanisms of transcript elongation and its regulation.
S. M. Uptain (1997)
10.1101/GAD.3.12B.2202
Flexible recognition of rapidly evolving promoter sequences by mitochondrial transcription factor 1.
R. P. Fisher (1989)



This paper is referenced by
10.2210/pdb3tmm/pdb
TFAM imposes a U-turn on mitochondrial DNA
Huu B. Ngo (2011)
10.1073/pnas.0910581107
Core human mitochondrial transcription apparatus is a regulated two-component system in vitro
T. Shutt (2010)
Structure and function of human mitochondrial RNA polymerase elongation complex.
Kathrin Schwinghammer (2014)
10.1093/nar/gks565
Human Cockayne syndrome B protein reciprocally communicates with mitochondrial proteins and promotes transcriptional elongation
B. Berquist (2012)
10.1080/21541264.2017.1331156
Topological requirements of the mitochondrial heavy-strand promoters
Ornella Zollo (2017)
10.1201/B16512-5
Forensic Aspects of mtDNA Analysis
M. Holland (2014)
10.1016/j.theriogenology.2019.05.001
Expression of selected mitochondrial genes during in vitro maturation of bovine oocytes related to their meiotic competence.
L. Nemcova (2019)
10.1002/pro.3688
Mechanisms of mammalian mitochondrial transcription
Emilie Bouda (2019)
10.1016/j.bbagrm.2012.06.002
Mitochondrial DNA damage and its consequences for mitochondrial gene expression.
Susan D. Cline (2012)
10.1038/nsmb.2159
Tfam, a mitochondrial transcription and packaging factor, imposes a U-turn on mitochondrial DNA
Huu B. Ngo (2011)
10.17760/d20260458
Single molecule studies of DNA bending, looping and compacting proteins using optical tweezers and atomic force microscopy
Divakaran Murugesapillai (2017)
10.1007/978-1-4939-3040-1_15
Expression and Purification of Mitochondrial RNA Polymerase and Transcription Factor A from Drosophila melanogaster.
John P. Gajewski (2016)
10.1073/pnas.1108852108
Mitochondrial Ribosomal Protein L12 selectively associates with human mitochondrial RNA polymerase to activate transcription
Y. Surovtseva (2011)
10.1128/AAC.02351-13
Inhibition of Hepatitis C Virus Replication by GS-6620, a Potent C-Nucleoside Monophosphate Prodrug
J. Feng (2014)
10.1021/bi300074j
Conservation of promoter melting mechanisms in divergent regions of the single-subunit RNA polymerases.
G. Velázquez (2012)
10.1007/s10555-018-9772-7
Roles of the mitochondrial genetics in cancer metastasis: not to be ignored any longer
T. Beadnell (2018)
10.1016/j.antiviral.2017.04.005
Structure‐activity relationship analysis of mitochondrial toxicity caused by antiviral ribonucleoside analogs
Zhinan Jin (2017)
10.1371/journal.ppat.1003030
Sensitivity of Mitochondrial Transcription and Resistance of RNA Polymerase II Dependent Nuclear Transcription to Antiviral Ribonucleosides
J. Arnold (2012)
10.1016/j.bbagrm.2015.05.010
Structural models of mammalian mitochondrial transcription factor B2.
I. Moustafa (2015)
10.1093/nar/gkq656
Arrest of human mitochondrial RNA polymerase transcription by the biological aldehyde adduct of DNA, M1dG
Susan D. Cline (2010)
10.1073/pnas.1118710109
Transcription from the second heavy-strand promoter of human mtDNA is repressed by transcription factor A in vitro
M. F. Lodeiro (2012)
10.1093/nar/gkr787
Transcriptional activation by mitochondrial transcription factor A involves preferential distortion of promoter DNA
C. Malarkey (2012)
10.7554/eLife.27283
Unexpected sequences and structures of mtDNA required for efficient transcription from the first heavy-strand promoter
A. Uchida (2017)
10.1201/B19420-6
Transcriptional Regulation of Mitochondrial Biogenesis and Quality Control
H. Suliman (2015)
10.1146/annurev-biochem-060815-014402
Maintenance and Expression of Mammalian Mitochondrial DNA.
C. Gustafsson (2016)
10.2741/4520
Mitochondrial transcription in mammalian cells.
I. Shokolenko (2017)
10.1038/nature17180
Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys
T. Warren (2016)
10.1016/j.bbagrm.2012.04.002
Human mitochondrial RNA polymerase: structure-function, mechanism and inhibition.
J. Arnold (2012)
10.1016/j.tibs.2013.03.006
Accessorizing the human mitochondrial transcription machinery.
Megan Bestwick (2013)
10.1021/bi200350d
Human mitochondrial RNA polymerase: evaluation of the single-nucleotide-addition cycle on synthetic RNA/DNA scaffolds.
Eric D. Smidansky (2011)
10.1007/978-3-642-22380-8_11
Mechanism and Regulation of Mitochondrial Transcription in Animal Cells
Paola Loguercio Polosa (2012)
10.1101/129908
Unexpected sequences and structures of mtDNA required for efficient transcription from the first heavy-strand promoter
A. Uchida (2017)
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