A novel intermediate in transcription initiation by human mitochondrial RNA polymerase

Nucleic Acids Research, Apr 2014

The mitochondrial genome is transcribed by a single-subunit T7 phage-like RNA polymerase (mtRNAP), structurally unrelated to cellular RNAPs. In higher eukaryotes, mtRNAP requires two transcription factors for efficient initiation—TFAM, a major nucleoid protein, and TFB2M, a transient component of mtRNAP catalytic site. The mechanisms behind assembly of the mitochondrial transcription machinery and its regulation are poorly understood. We isolated and identified a previously unknown human mitochondrial transcription intermediate—a pre-initiation complex that includes mtRNAP, TFAM and promoter DNA. Using protein–protein cross-linking, we demonstrate that human TFAM binds to the N-terminal domain of mtRNAP, which results in bending of the promoter DNA around mtRNAP. The subsequent recruitment of TFB2M induces promoter melting and formation of an open initiation complex. Our data indicate that the pre-initiation complex is likely to be an important target for transcription regulation and provide basis for further structural, biochemical and biophysical studies of mitochondrial transcription.

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A novel intermediate in transcription initiation by human mitochondrial RNA polymerase

Yaroslav I. Morozov 1 2 Karen Agaronyan 1 2 Alan C. M. Cheung 0 1 Michael Anikin 1 2 Patrick Cramer 0 1 Dmitry Temiakov 1 2 0 Gene Center and Department of Biochemistry, Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universita t M unchen , Feodor-Lynen-Strasse 25, 81377 Munich, Germany 1 Present address: Alan C. M. Cheung, Institute of Structural and Molecular Biology, University College London/Birkbeck , Malet Street, London WC1E 7HX, UK 2 Department of Cell Biology, School of Osteopathic Medicine, Rowan University, Medical Center Dr , Stratford, NJ 08084, USA The mitochondrial genome is transcribed by a single-subunit T7 phage-like RNA polymerase (mtRNAP), structurally unrelated to cellular RNAPs. In higher eukaryotes, mtRNAP requires two transcription factors for efficient initiationTFAM, a major nucleoid protein, and TFB2M, a transient component of mtRNAP catalytic site. The mechanisms behind assembly of the mitochondrial transcription machinery and its regulation are poorly understood. We isolated and identified a previously unknown human mitochondrial transcription intermediatea pre-initiation complex that includes mtRNAP, TFAM and promoter DNA. Using proteinprotein cross-linking, we demonstrate that human TFAM binds to the N-terminal domain of mtRNAP, which results in bending of the promoter DNA around mtRNAP. The subsequent recruitment of TFB2M induces promoter melting and formation of an open initiation complex. Our data indicate that the pre-initiation complex is likely to be an important target for transcription regulation and provide basis for further structural, biochemical and biophysical studies of mitochondrial transcription. - INTRODUCTION Mitochondrial transcription does not fit the paradigm of eukaryotic or prokaryotic transcription systems, as it relies on a single subunit T7 phage-like mtRNAP. However, unlike the T7 system, transcription initiation by mtRNAP involves a number of additional factors, suggesting a more complex organization that likely reflects regulatory needs by the mitochondrial system (1,2). Cellular multi-subunit polymerases form an array of transient complexes along the pathway to transcription initiation (37). These intermediates serve as important targets for regulation by presenting a specific conformation of RNAP to various regulatory factors. Binding of these factors at early stages of transcription is an important mechanism that affects cellular physiology and development, and this phenomenon has been well studied in a number of systems (8,9). As noted above, mtRNAP also requires auxiliary factors for transcription initiation; however, the mechanisms of promoter recognition, binding and melting by the mtRNAP must be distinct from those established for phage T7 RNAP (1012), which does not require such factors, and in which formation of stable transcription intermediates has not been reported (13,14). While most eukaryotic organisms contain mitochondria, the basal mitochondrial transcription machinery appears to have evolved differently in lower and higher eukaryotes. Thus, the yeast core transcription system is composed of mtRNAP and a single transcription initiation factor, Mtf1, which is implicated in promoter melting (15,16). In contrast, the mammalian core transcription apparatus contains, in addition to mtRNAP and TFB2M (a functional analog of Mtf1), an abundant mitochondrial protein, TFAM (17,18) that is a major component of the mitochondrial nucleoid and is required for mtDNA organization and maintenance; knockout of the latter protein results in a dramatic loss of mtDNA and disruption of oxidative phosphorylation (19,20). While yeast mitochondria also contain TFAM, it has no apparent role in transcription and serves only as a nucleoid protein, likely due to truncation (as compared with human TFAM) of a C-terminal tail domain that has been implicated in transcription activation in human mitochondria (21). Human TFB2M is transiently associated with mtRNAP during initiation and interacts with the templating DNA base and the priming substrate (22). Both TFB2M and Mtf1 have been implicated in regulation of transcription initiation in response to variations in cellular ATP concentrations (22,23). Despite recent progress in structural studies of human mtRNAP and TFAMDNA complexes (2,2426), the mechanisms of assembly of the mitochondrial transcription initiation complex are poorly understood and are somewhat controversial. It has been suggested that TFAM, which leaves a clear footprint on two major human mitochondrial promoters, termed LSP and HSP1 (21,27), interacts via its C-terminus with TFB2M, implicating the latter in recruiting mtRNAP to its promoter (28). On the other hand, it has been postulated that the mitochondrial core transcription system includes only mtRNAP and TFB2M, and that TFAM is dispensable for the initiation process and acts to stimulate basal levels of transcription from both the LSP and HSP1 promoters (29) (...truncated)


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Yaroslav I. Morozov, Karen Agaronyan, Alan C. M. Cheung, Michael Anikin, Patrick Cramer, Dmitry Temiakov. A novel intermediate in transcription initiation by human mitochondrial RNA polymerase, Nucleic Acids Research, 2014, pp. 3884-3893, 42/6, DOI: 10.1093/nar/gkt1356