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TEFM is a potent stimulator of mitochondrial transcription elongation in vitro

Journal article
Authors Viktor Posse
S. Shahzad
Maria Falkenberg
B. M. Hallberg
Claes M Gustafsson
Published in Nucleic Acids Research
Volume 43
Issue 5
Pages 2615-2624
ISSN 0305-1048
Publication year 2015
Published at Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology
Pages 2615-2624
Language en
Links dx.doi.org/10.1093/nar/gkv105
Keywords RNA-DNA HYBRID, PRIMER FORMATION, HUMAN MTDNA, POLYMERASE, REPLICATION, PROMOTER, TERMINATION, INITIATION, COMPONENT, SEQUENCE, Biochemistry & Molecular Biology, ATES OF AMERICA, V107, P16072
Subject categories Biochemistry and Molecular Biology

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.

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