Biophysical characterizations of human mitochondrial transcription factor A and its binding to tumor suppressor p53 (original) (raw)
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Nucleic Acids Research, 2009
The mitochondrial transcription factor A (mtTFA) is central to assembly and initiation of the mitochondrial transcription complex. Human mtTFA (h-mtTFA) is a dual high mobility group box (HMGB) protein that binds site-specifically to the mitochondrial genome and demarcates the promoters for recruitment of h-mtTFB1, h-mtTFB2 and the mitochondrial RNA polymerase. The stoichiometry of h-mtTFA was found to be a monomer in the absence of DNA, whereas it formed a dimer in the complex with the light strand promoter (LSP) DNA. Each of the HMG boxes and the C-terminal tail were evaluated for their ability to bind to the LSP DNA. Removal of the C-terminal tail only slightly decreased nonsequence specific DNA binding, and box A, but not box B, was capable of binding to the LSP DNA. The X-ray crystal structure of h-mtTFA box B, at 1.35 Å resolution, revealed the features of a noncanonical HMG box. Interactions of box B with other regions of h-mtTFA were observed. Together, these results provide an explanation for the unusual DNA-binding properties of box B and suggest possible roles for this domain in transcription complex assembly.
Nucleic Acids Research, 2019
Human mitochondrial DNA (h-mtDNA) codes for 13 subunits of the oxidative phosphorylation pathway, the essential route that produces ATP. H-mtDNA transcription and replication depends on the transcription factor TFAM, which also maintains and compacts this genome. It is well-established that TFAM activates the mtDNA promoters LSP and HSP1 at the mtDNA control region where DNA regulatory elements cluster. Previous studies identified still uncharacterized, additional binding sites at the control region downstream from and slightly similar to LSP, namely sequences X and Y (Site-X and Site-Y) (Fisher et al., Cell 50, pp 247–258, 1987). Here, we explore TFAM binding at these two sites and compare them to LSP by multiple experimental and in silico methods. Our results show that TFAM binding is strongly modulated by the sequence-dependent properties of Site-X, Site-Y and LSP. The high binding versatility of Site-Y or the considerable stiffness of Site-X tune TFAM interactions. In addition, ...
Genes to cells : devoted to molecular & cellular mechanisms, 2015
Mitochondrial transcription factor A (TFAM) is a key regulator of mitochondrial DNA (mtDNA). TFAM interacts with itself and forms dimers; however, the precise interaction domain in vivo has not yet been determined. We herein showed that human TFAM formed oligomers in mitochondria by in situ chemical cross-linking. We used the separated fluorescent protein, monomeric Kusabira-Green, as a reporter to monitor their self-association in mitochondria. This reporter successfully detected the TFAM-TFAM interaction in cells as fluorescent signals on mitochondria. We also found that the N-terminal high-mobility group box domain was sufficient for this interaction. The expression of the dimer-defective mutant induced enlarged mtDNA nucleoids, suggesting the importance of dimerization in the distribution of mtDNA. The reporter system also supported the association and mixture between independent nucleoids through TFAM by a cell fusion assay using hemagglutinating virus of Japan. We here, for th...
Proceedings of the National Academy of Sciences, 2012
The tumor suppressor p53 is a homotetramer of 4 × 393 residues. Its core domain and tetramerization domain are linked and flanked by intrinsically disordered sequences, which hinder its full structural characterization. There is an outstanding problem of the state of the tetramerization domain. Structural studies on the isolated tetramerization domain show it is in a folded tetrameric conformation, but there are conflicting models from electron microscopy of the full-length protein, one of which proposes that the domain is not tetramerically folded and the tetrameric protein is stabilized by interactions between the N and C termini. Here, we present methyl-transverse relaxation optimized NMR spectroscopy (methyl-TROSY) investigations on the full-length and separate domains of the protein with its methionine residues enriched with 13 C to probe its quaternary structure. We obtained high-quality spectra of both the full-length tetrameric p53 and its DNA complex, observing the environment at 11 specific methyl sites. The tetramerization domain was as tetramerically folded in the full-length constructs as in the isolated domain. The N and C termini were intrinsically disordered in both the full-length protein and its complex with a 20-residue specific DNA sequence. Additionally, we detected in the interface of the core (DNA-binding) and N-terminal parts of the protein a slow conformational exchange process that was modulated by specific recognition of DNA, indicating allosteric processes.
Architectural Role of Mitochondrial Transcription Factor A in Maintenance of Human Mitochondrial DNA
Molecular and Cellular Biology, 2004
Mitochondrial transcription factor A (TFAM), a transcription factor for mitochondrial DNA (mtDNA) that also possesses the property of nonspecific DNA binding, is essential for maintenance of mtDNA. To clarify the role of TFAM, we repressed the expression of endogenous TFAM in HeLa cells by RNA interference. The amount of TFAM decreased maximally to about 15% of the normal level at
Recognition of DNA by p53 Core Domain and Location of Intermolecular Contacts of Cooperative Binding
Journal of Molecular Biology, 2002
We present an analysis by NMR of a 58 kDa complex of the core domain of the tumour suppressor p53 with DNA that complements and extends the crystal structure analysis. Binding of specific DNA caused significant chemical shifts of residues on the DNA-binding interface that translated into the b-sheet of the protein. Binding of non-specific DNA caused weak but qualitatively the same shifts, corresponding to weaker binding interactions. The observed chemical shift differences correlate with frequency of cancer-inducing mutations, suggesting that the affected residues contribute to the stability of p53 core domain-DNA complex. We also identified two affected regions on the surface of the protein: helix 1 (residues V173 -C182) plus G244 and residues L114-T118, which may represent a dimerisation interface.
Regulation of DNA Binding of p53 by its C-terminal Domain
Journal of Molecular Biology, 2004
The tumor suppressor p53 is a tetrameric multi-domain transcription factor. Its C-terminal domain is thought to regulate the binding of its core domain to specific recognition sequences in promoters. The mechanism of regulation by the C-terminal domain and the role of its post-translational modification are controversial. We have examined the binding of DNA in solution to a series of unmodified p53 constructs that lack various domains. The specific DNA sequences bind tightly to the core domain, irrespective of whether or not the C-terminal domain is part of the construct. Unmodified p53 is accordingly an active DNA binding protein. Non-specific DNA sequences do not inhibit directly the binding of the specific sequences to the core but bind to the C terminus and inhibit p53 via that binding mode. Using NMR, we identified the residues of the C terminus that interact with the non-specific DNA. They include residues that are known to be modified post-translationally. Our data provide direct support for the regulatory role of the C terminus in the activity of p53 and show that p53 containing the unmodified C terminus actively binds to short double-stranded DNA.