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TEFM is a potent stimulator of mitochondrial transcription elongation in vitro

A single-subunit RNA polymerase, POLRMT, transcribes the mitochondrial genome in human cells. Recently, a factor termed as the mitochondrial transcription elongation factor, TEFM, was shown to stimulate transcription elongation in vivo, but its effect in vitro was relatively modest. In the current w...

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Autores principales: Posse, Viktor, Shahzad, Saba, Falkenberg, Maria, Hällberg, B. Martin, Gustafsson, Claes M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4357710/
https://www.ncbi.nlm.nih.gov/pubmed/25690892
http://dx.doi.org/10.1093/nar/gkv105
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author Posse, Viktor
Shahzad, Saba
Falkenberg, Maria
Hällberg, B. Martin
Gustafsson, Claes M.
author_facet Posse, Viktor
Shahzad, Saba
Falkenberg, Maria
Hällberg, B. Martin
Gustafsson, Claes M.
author_sort Posse, Viktor
collection PubMed
description A single-subunit RNA polymerase, POLRMT, transcribes the mitochondrial genome in human cells. Recently, a factor termed as the mitochondrial transcription elongation factor, TEFM, was shown to stimulate transcription elongation in vivo, but its effect in vitro was relatively modest. In the current work, we have isolated active TEFM in recombinant form and used a reconstituted in vitro transcription system to characterize its activities. We show that TEFM strongly promotes POLRMT processivity as it dramatically stimulates the formation of longer transcripts. TEFM also abolishes premature transcription termination at conserved sequence block II, an event that has been linked to primer formation during initiation of mtDNA synthesis. We show that POLRMT pauses at a wide range of sites in a given DNA sequence. In the absence of TEFM, this leads to termination; however, the presence of TEFM abolishes this effect and aids POLRMT in continuation of transcription. Further, we show that TEFM substantially increases the POLRMT affinity to an elongation-like DNA:RNA template. In combination with previously published in vivo observations, our data establish TEFM as an essential component of the mitochondrial transcription machinery.
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spelling pubmed-43577102015-03-20 TEFM is a potent stimulator of mitochondrial transcription elongation in vitro Posse, Viktor Shahzad, Saba Falkenberg, Maria Hällberg, B. Martin Gustafsson, Claes M. Nucleic Acids Res Gene regulation, Chromatin and Epigenetics A single-subunit RNA polymerase, POLRMT, transcribes the mitochondrial genome in human cells. Recently, a factor termed as the mitochondrial transcription elongation factor, TEFM, was shown to stimulate transcription elongation in vivo, but its effect in vitro was relatively modest. In the current work, we have isolated active TEFM in recombinant form and used a reconstituted in vitro transcription system to characterize its activities. We show that TEFM strongly promotes POLRMT processivity as it dramatically stimulates the formation of longer transcripts. TEFM also abolishes premature transcription termination at conserved sequence block II, an event that has been linked to primer formation during initiation of mtDNA synthesis. We show that POLRMT pauses at a wide range of sites in a given DNA sequence. In the absence of TEFM, this leads to termination; however, the presence of TEFM abolishes this effect and aids POLRMT in continuation of transcription. Further, we show that TEFM substantially increases the POLRMT affinity to an elongation-like DNA:RNA template. In combination with previously published in vivo observations, our data establish TEFM as an essential component of the mitochondrial transcription machinery. Oxford University Press 2015-03-11 2015-02-17 /pmc/articles/PMC4357710/ /pubmed/25690892 http://dx.doi.org/10.1093/nar/gkv105 Text en © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Gene regulation, Chromatin and Epigenetics
Posse, Viktor
Shahzad, Saba
Falkenberg, Maria
Hällberg, B. Martin
Gustafsson, Claes M.
TEFM is a potent stimulator of mitochondrial transcription elongation in vitro
title TEFM is a potent stimulator of mitochondrial transcription elongation in vitro
title_full TEFM is a potent stimulator of mitochondrial transcription elongation in vitro
title_fullStr TEFM is a potent stimulator of mitochondrial transcription elongation in vitro
title_full_unstemmed TEFM is a potent stimulator of mitochondrial transcription elongation in vitro
title_short TEFM is a potent stimulator of mitochondrial transcription elongation in vitro
title_sort tefm is a potent stimulator of mitochondrial transcription elongation in vitro
topic Gene regulation, Chromatin and Epigenetics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4357710/
https://www.ncbi.nlm.nih.gov/pubmed/25690892
http://dx.doi.org/10.1093/nar/gkv105
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