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Human Mitochondrial Transcription Factor B2 Is Required for Promoter Melting during Initiation of Transcription

Journal article
Authors Viktor Posse
Claes M Gustafsson
Published in Journal of Biological Chemistry
Volume 292
Issue 7
Pages 2637-2645
ISSN 0021-9258
Publication year 2017
Published at Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology
Pages 2637-2645
Language en
Links doi.org/10.1074/jbc.M116.751008
Keywords rna-polymerase, in-vitro, dna, tfam, specificity, complexes, component, binding, protein, light, Biochemistry & Molecular Biology
Subject categories Biochemistry and Molecular Biology

Abstract

The mitochondrial transcription initiation machinery in humans consists of three proteins: the RNA polymerase (POLRMT) and two accessory factors, transcription factors A and B2 (TFAM and TFB2M, respectively). This machinery is required for the expression of mitochondrial DNA and the biogenesis of the oxidative phosphorylation system. Previous experiments suggested that TFB2M is required for promoter melting, but conclusive experimental proof for this effect has not been presented. Moreover, the role of TFB2M in promoter unwinding has not been discriminated from that of TFAM. Here we used potassium permanganate footprinting, DNase I footprinting, and in vitro transcription from the mitochondrial light-strand promoter to study the role of TFB2M in transcription initiation. We demonstrate that a complex composed of TFAM and POLRMT was readily formed at the promoter but alone was insufficient for promoter melting, which only occurred when TFB2M joined the complex. We also show that mismatch bubble templates could circumvent the requirement of TFB2M, but TFAM was still required for efficient initiation. Our findings support a model in which TFAM first recruits POLRMT to the promoter, followed by TFB2M binding and induction of promoter melting.

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