Transcription from the second heavy-strand promoter of human mtDNA is repressed by transcription factor A in vitro

Maria F. Lodeiro, Akira Uchida, Megan Bestwick, Ibrahim Moustafa, Jamie Jon Arnold, Gerald S. Shadel, Craig Eugene Cameron

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Abstract

Cell-based studies support the existence of two promoters on the heavy strand of mtDNA: heavy-strand promoter 1 (HSP1) and HSP2. However, transcription from HSP2 has been reported only once in a cell-free system, and never when recombinant proteins have been used. Here, we document transcription from HSP2 using an in vitro system of defined composition. An oligonucleotide template representing positions 596-685 of mtDNA was sufficient to observe transcription by the human mtRNA polymerase (POLRMT) that was absolutely dependent on mitochondrial transcription factor B2 (TFB2M). POLRMT/TFB2M-dependent transcription was inhibited by concentrations of mitochondrial transcription factor A (TFAM) stoichiometric with the transcription template, a condition that activates transcription from the light-strand promoter (LSP) in vitro. Domains of TFAM required for LSP activation were also required for HSP2 repression, whereas other mtDNA binding proteins failed to alter transcriptional output. Binding sites for TFAM were located on both sides of the start site of transcription from HSP2, suggesting that TFAM binding interferes with POLRMT and/or TFB2M binding. Consistent with a competitive bindingmodel for TFAMrepression of HSP2, the impact of TFAM concentration on HSP2 transcription was diminished by elevating the POLRMT and TFB2M concentrations. In the context of our previous studies of LSP and HSP1, it is now clear that three promoters exist in human mtDNA. Each promoter has a unique requirement for and/or response to the level of TFAM present, thus implying far greater complexity in the regulation of mammalian mitochondrial transcription than recognized to date.

Original languageEnglish (US)
Pages (from-to)6513-6518
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume109
Issue number17
DOIs
StatePublished - Apr 24 2012

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Mitochondrial DNA
Transcription Factors
Light
Cell-Free System
Transcription Initiation Site
Recombinant Proteins
Oligonucleotides
Carrier Proteins
Binding Sites
In Vitro Techniques

All Science Journal Classification (ASJC) codes

  • General

Cite this

Lodeiro, Maria F. ; Uchida, Akira ; Bestwick, Megan ; Moustafa, Ibrahim ; Arnold, Jamie Jon ; Shadel, Gerald S. ; Cameron, Craig Eugene. / Transcription from the second heavy-strand promoter of human mtDNA is repressed by transcription factor A in vitro. In: Proceedings of the National Academy of Sciences of the United States of America. 2012 ; Vol. 109, No. 17. pp. 6513-6518.
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Transcription from the second heavy-strand promoter of human mtDNA is repressed by transcription factor A in vitro. / Lodeiro, Maria F.; Uchida, Akira; Bestwick, Megan; Moustafa, Ibrahim; Arnold, Jamie Jon; Shadel, Gerald S.; Cameron, Craig Eugene.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 109, No. 17, 24.04.2012, p. 6513-6518.

Research output: Contribution to journalArticle

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T1 - Transcription from the second heavy-strand promoter of human mtDNA is repressed by transcription factor A in vitro

AU - Lodeiro, Maria F.

AU - Uchida, Akira

AU - Bestwick, Megan

AU - Moustafa, Ibrahim

AU - Arnold, Jamie Jon

AU - Shadel, Gerald S.

AU - Cameron, Craig Eugene

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N2 - Cell-based studies support the existence of two promoters on the heavy strand of mtDNA: heavy-strand promoter 1 (HSP1) and HSP2. However, transcription from HSP2 has been reported only once in a cell-free system, and never when recombinant proteins have been used. Here, we document transcription from HSP2 using an in vitro system of defined composition. An oligonucleotide template representing positions 596-685 of mtDNA was sufficient to observe transcription by the human mtRNA polymerase (POLRMT) that was absolutely dependent on mitochondrial transcription factor B2 (TFB2M). POLRMT/TFB2M-dependent transcription was inhibited by concentrations of mitochondrial transcription factor A (TFAM) stoichiometric with the transcription template, a condition that activates transcription from the light-strand promoter (LSP) in vitro. Domains of TFAM required for LSP activation were also required for HSP2 repression, whereas other mtDNA binding proteins failed to alter transcriptional output. Binding sites for TFAM were located on both sides of the start site of transcription from HSP2, suggesting that TFAM binding interferes with POLRMT and/or TFB2M binding. Consistent with a competitive bindingmodel for TFAMrepression of HSP2, the impact of TFAM concentration on HSP2 transcription was diminished by elevating the POLRMT and TFB2M concentrations. In the context of our previous studies of LSP and HSP1, it is now clear that three promoters exist in human mtDNA. Each promoter has a unique requirement for and/or response to the level of TFAM present, thus implying far greater complexity in the regulation of mammalian mitochondrial transcription than recognized to date.

AB - Cell-based studies support the existence of two promoters on the heavy strand of mtDNA: heavy-strand promoter 1 (HSP1) and HSP2. However, transcription from HSP2 has been reported only once in a cell-free system, and never when recombinant proteins have been used. Here, we document transcription from HSP2 using an in vitro system of defined composition. An oligonucleotide template representing positions 596-685 of mtDNA was sufficient to observe transcription by the human mtRNA polymerase (POLRMT) that was absolutely dependent on mitochondrial transcription factor B2 (TFB2M). POLRMT/TFB2M-dependent transcription was inhibited by concentrations of mitochondrial transcription factor A (TFAM) stoichiometric with the transcription template, a condition that activates transcription from the light-strand promoter (LSP) in vitro. Domains of TFAM required for LSP activation were also required for HSP2 repression, whereas other mtDNA binding proteins failed to alter transcriptional output. Binding sites for TFAM were located on both sides of the start site of transcription from HSP2, suggesting that TFAM binding interferes with POLRMT and/or TFB2M binding. Consistent with a competitive bindingmodel for TFAMrepression of HSP2, the impact of TFAM concentration on HSP2 transcription was diminished by elevating the POLRMT and TFB2M concentrations. In the context of our previous studies of LSP and HSP1, it is now clear that three promoters exist in human mtDNA. Each promoter has a unique requirement for and/or response to the level of TFAM present, thus implying far greater complexity in the regulation of mammalian mitochondrial transcription than recognized to date.

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