title: TEFM is a potent stimulator of mitochondrial transcription elongation in vitro.
abstract: 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.
authors.affiliation: Department of Medical Biochemistry and Cell Biology, University of Gothenburg, SE-40530 Gothenburg, Sweden.
authors.forename: Viktor
authors.initials: V
authors.lastname: Posse
authors.affiliation: Department of Cell and Molecular Biology, Karolinska Institutet, SE-17177 Stockholm, Sweden Röntgen-Ångström-Cluster, Karolinska Institutet Outstation, Centre for Structural Systems Biology, DESY-Campus, D-22603 Hamburg, Germany.
authors.forename: Saba
authors.initials: S
authors.lastname: Shahzad
authors.affiliation: Department of Medical Biochemistry and Cell Biology, University of Gothenburg, SE-40530 Gothenburg, Sweden.
authors.forename: Maria
authors.initials: M
authors.lastname: Falkenberg
authors.affiliation: Department of Cell and Molecular Biology, Karolinska Institutet, SE-17177 Stockholm, Sweden Röntgen-Ångström-Cluster, Karolinska Institutet Outstation, Centre for Structural Systems Biology, DESY-Campus, D-22603 Hamburg, Germany European Molecular Biology Laboratory, Hamburg Unit, D-22603 Hamburg, Germany.
authors.forename: B Martin
authors.initials: BM
authors.lastname: Hällberg
authors.affiliation: Department of Medical Biochemistry and Cell Biology, University of Gothenburg, SE-40530 Gothenburg, Sweden claes.gustafsson@medkem.gu.se.
authors.forename: Claes M
authors.initials: CM
authors.lastname: Gustafsson
mesh_terms: D000080242:8-Hydroxy-2'-Deoxyguanosine; D002474:Cell-Free System; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D004272:DNA, Mitochondrial / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism; D054629:Genome, Mitochondrial / Q000235:genetics; D006801:Humans; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D008957:Models, Genetic; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013698:Templates, Genetic; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D004272:DNA, Mitochondrial; D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; C560090:TEFM protein, human; D014157:Transcription Factors; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D012321:DNA-Directed RNA Polymerases; C519567:POLRMT protein, human; D003849:Deoxyguanosine