Structural analysis and DNA binding of the HMG domains of the human mitochondrial transcription factor A

Gangelhoff, Todd A.; Mungalachetty, Purnima S.; Nix, Jay C.; Churchill, Mair E. A.

doi:10.1093/nar/gkp157

Cited by 88 publications

(128 citation statements)

References 54 publications

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“…S1 in the supplemental material). Also, the observation that the centrally located HMG box, rather than the C-terminal HMG box, seems to play a dominant role in DNA binding is consistent with the properties of both ScAbf2p (84) and human TFAM (85). The possibility that in addition to the similarities with other mtHMG proteins, YlMhb1p may also possess some unique properties will be assessed in more detail in our further studies.…”

Section: Discussionmentioning

confidence: 59%

The Strictly Aerobic Yeast Yarrowia lipolytica Tolerates Loss of a Mitochondrial DNA-Packaging Protein

et al. 2014

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bMitochondrial DNA (mtDNA) is highly compacted into DNA-protein structures termed mitochondrial nucleoids (mt-nucleoids). The key mt-nucleoid components responsible for mtDNA condensation are HMG box-containing proteins such as mammalian mitochondrial transcription factor A (TFAM) and Abf2p of the yeast Saccharomyces cerevisiae. To gain insight into the function and organization of mt-nucleoids in strictly aerobic organisms, we initiated studies of these DNA-protein structures in Yarrowia lipolytica. We identified a principal component of mt-nucleoids in this yeast and termed it YlMhb1p (Y. lipolytica mitochondrial HMG box-containing protein 1). YlMhb1p contains two putative HMG boxes contributing both to DNA binding and to its ability to compact mtDNA in vitro. Phenotypic analysis of a ⌬mhb1 strain lacking YlMhb1p resulted in three interesting findings. First, although the mutant exhibits clear differences in mt-nucleoids accompanied by a large decrease in the mtDNA copy number and the number of mtDNA-derived transcripts, its respiratory characteristics and growth under most of the conditions tested are indistinguishable from those of the wild-type strain. Second, our results indicate that a potential imbalance between subunits of the respiratory chain encoded separately by nuclear DNA and mtDNA is prevented at a (post)translational level. Third, we found that mtDNA in the ⌬mhb1 strain is more prone to mutations, indicating that mtHMG box-containing proteins protect the mitochondrial genome against mutagenic events. Individual eukaryotic cells contain a population of mitochondrial DNA (mtDNA) molecules, the number of which may reach several thousand copies. For example, aerobically grown diploid cells of the yeast Saccharomyces cerevisiae contain, on average, 100 molecules of 85-kbp mtDNA. With a distance of 0.34 nm between base pairs in DNA, the total length of mtDNA reaches almost 3 mm per cell, while the diameter of the cell does not exceed 3 to 4 m. Analogously to its nuclear counterpart, mtDNA must be packaged into condensed nucleoprotein structures termed mitochondrial nucleoids (mt-nucleoids) (1-6). The size of mt-nucleoids in S. cerevisiae ranges from 0.2 to 0.9 m, meaning that mtDNA in yeasts undergoes compaction of roughly 3 orders of magnitude. Although it is known that the size and shape (oval versus globular) of mt-nucleoids, the number of mt-nucleoids (ranging from 50 to 70) per diploid cell (2), and the number of copies (up to 9) of mtDNA per mt-nucleoid (3) in S. cerevisiae depend on physiological conditions, the molecular mechanisms mediating nucleoid maintenance, dynamics, and roles in mtDNA distribution/segregation during cell division are largely not understood. This is also true for mammalian mt-nucleoids, which, in contrast to their yeast counterparts, contain a relatively small number of mtDNA molecules and are thus more solitary in nature (7,8). Description of these intra-and interspecific differences in the organization of mt-nucleoids would greatly facilitate our understanding of the m...

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Section: Discussionmentioning

confidence: 59%

The Strictly Aerobic Yeast Yarrowia lipolytica Tolerates Loss of a Mitochondrial DNA-Packaging Protein

et al. 2014

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“…The CTD seems to be uniquely important for the transcriptional functions of TFAM (8). Portions of the CTD seem to govern DNA binding specificity/affinity (5,19,20), and portions seem to govern interactions with proteins (for example, TFB2M) (3). Therefore, we asked if the CTD was important for transcriptional repression of HSP2 by TFAM.…”

Section: Resultsmentioning

confidence: 99%

Transcription from the second heavy-strand promoter of human mtDNA is repressed by transcription factor A in vitro

Lodeiro

Uchida

Bestwick

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Cell-based studies support the existence of two promoters on the heavy strand of mtDNA: heavy-strand promoter 1 (HSP1) and HSP2. However, transcription from HSP2 has been reported only once in a cell-free system, and never when recombinant proteins have been used. Here, we document transcription from HSP2 using an in vitro system of defined composition. An oligonucleotide template representing positions 596-685 of mtDNA was sufficient to observe transcription by the human mtRNA polymerase (POLRMT) that was absolutely dependent on mitochondrial transcription factor B2 (TFB2M). POLRMT/TFB2M-dependent transcription was inhibited by concentrations of mitochondrial transcription factor A (TFAM) stoichiometric with the transcription template, a condition that activates transcription from the light-strand promoter (LSP) in vitro. Domains of TFAM required for LSP activation were also required for HSP2 repression, whereas other mtDNA binding proteins failed to alter transcriptional output. Binding sites for TFAM were located on both sides of the start site of transcription from HSP2, suggesting that TFAM binding interferes with POLRMT and/or TFB2M binding. Consistent with a competitive binding model for TFAM repression of HSP2, the impact of TFAM concentration on HSP2 transcription was diminished by elevating the POLRMT and TFB2M concentrations. In the context of our previous studies of LSP and HSP1, it is now clear that three promoters exist in human mtDNA. Each promoter has a unique requirement for and/or response to the level of TFAM present, thus implying far greater complexity in the regulation of mammalian mitochondrial transcription than recognized to date. mitochondria | gene expression | initiation | gel-shift assay | footprinting

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“…Although the mechanisms by which Lon recognizes its target proteins are not well understood, a recent report showed that E. coli Lon recognizes specific aromatic residue-rich sequences that are hidden in the hydrophobic cores of native structures, but are accessible in unfolded structures (42). Interestingly, the HMG boxes in TFAM contain four conserved aromatic residues within a hydrophobic core, and these residues may be masked when TFAM binds DNA (25,27,29). Another possible explanation is that TFAM not bound to mtDNA becomes exposed to oxidative stress, whereas TFAM bound to mtDNA comprises part of the core of the mitochondrial nucleoids, and is thus surrounded by other proteins (43).…”

Section: Discussionmentioning

confidence: 99%

“…The major protein component of mtDNA nucleoids is mitochondrial transcription factor A (TFAM or mtTFA) (23,24). TFAM contains two high mobility group (HMG) amino acid sequence boxes; it binds to mtDNA both specifically and nonspecifically (25). TFAM is essential for mtDNA transcription and for mtDNA packaging in mtDNA maintenance (26)(27)(28)(29)(30).…”

mentioning

confidence: 99%

Mitochondrial Lon protease regulates mitochondrial DNA copy number and transcription by selective degradation of mitochondrial transcription factor A (TFAM)

Matsushima

Goto

Kaguni

2010

Proc. Natl. Acad. Sci. U.S.A.

250

220

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Lon is the major protease in the mitochondrial matrix in eukaryotes, and is well conserved among species. Although a role for Lon in mitochondrial biogenesis has been proposed, the mechanistic basis is unclear. Here, we demonstrate a role for Lon in mtDNA metabolism. An RNA interference (RNAi) construct was designed that reduces Lon to less than 10% of its normal level in Drosophila Schneider cells. RNAi knockdown of Lon results in increased abundance of mitochondrial transcription factor A (TFAM) and mtDNA copy number. In a corollary manner, overexpression of Lon reduces TFAM levels and mtDNA copy number. Notably, induction of mtDNA depletion in Lon knockdown cells does not result in degradation of TFAM, thereby causing a dramatic increase in the TFAM∶mtDNA ratio. The increased TFAM∶mtDNA ratio in turn causes inhibition of mitochondrial transcription. We conclude that Lon regulates mitochondrial transcription by stabilizing the mitochondrial TFAM∶ mtDNA ratio via selective degradation of TFAM.AAA+ protease | mtDNA maintenance | quality control

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Structural analysis and DNA binding of the HMG domains of the human mitochondrial transcription factor A

Cited by 88 publications

References 54 publications

The Strictly Aerobic Yeast Yarrowia lipolytica Tolerates Loss of a Mitochondrial DNA-Packaging Protein

The Strictly Aerobic Yeast Yarrowia lipolytica Tolerates Loss of a Mitochondrial DNA-Packaging Protein

Transcription from the second heavy-strand promoter of human mtDNA is repressed by transcription factor A in vitro

Mitochondrial Lon protease regulates mitochondrial DNA copy number and transcription by selective degradation of mitochondrial transcription factor A (TFAM)

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