Posse Viktor, Shahzad Saba, Falkenberg Maria, Hällberg B Martin, Gustafsson Claes M
Department of Medical Biochemistry and Cell Biology, University of Gothenburg, SE-40530 Gothenburg, Sweden.
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.
Nucleic Acids Res. 2015 Mar 11;43(5):2615-24. doi: 10.1093/nar/gkv105. Epub 2015 Feb 17.
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.
一种单亚基RNA聚合酶POLRMT负责转录人类细胞中的线粒体基因组。最近，一种被称为线粒体转录延伸因子（TEFM）的因子在体内被证明可刺激转录延伸，但其体外作用相对较小。在当前的工作中，我们以重组形式分离出了活性TEFM，并使用重组体外转录系统来表征其活性。我们发现，TEFM强烈促进POLRMT的持续合成能力，因为它能显著刺激更长转录本的形成。TEFM还消除了保守序列框II处的过早转录终止，这一事件与线粒体DNA合成起始过程中的引物形成有关。我们发现，POLRMT在给定DNA序列的广泛位点处暂停。在没有TEFM的情况下，这会导致终止；然而，TEFM的存在消除了这种影响，并帮助POLRMT继续转录。此外，我们发现TEFM显著增加了POLRMT对类似延伸的DNA:RNA模板的亲和力。结合之前发表的体内观察结果，我们的数据确定TEFM是线粒体转录机制的一个重要组成部分。
