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.
Highlights
The human mitochondrial genome is a circular DNA molecule of 16.6 kb that encodes 22 tRNAs, 2 rRNAs and 13 proteins required for oxidative phosphorylation
Recombinant TEFM interacts with POLRMT and drastically decreases pre-termination events
Subsequent analysis demonstrated that the knockdown of TEFM causes a decrease of promoter-distal mitochondrial transcripts, and affinity purification of TEFM brings down POLRMT and a number of mitochondrial transcription factors
Summary
The human mitochondrial genome (mtDNA) is a circular DNA molecule of 16.6 kb that encodes 22 tRNAs, 2 rRNAs and 13 proteins required for oxidative phosphorylation. Transcription of the genome is initiated from two regulatory sites in the control region of mtDNA, the heavy- and lightstrand promoters (HSP1 and LSP). In contrast to T7 RNAP, POLRMT cannot initiate promoter-dependent transcription on its own, as it needs two additional transcription factors, TFAM and TFB2M [1,8] These two factors are needed for the recruitment of POLRMT [9,10] and initial unwinding of the double-stranded promoter region [11], but they are not required for the transcription of a single-stranded DNA template or a template with a singlestranded DNA bubble covering the transcription start site [12]
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