Fig 5 - uploaded by Igor Antoshechkin
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Representative STEM images of xl-mtTFA tetramers bound to DNA. Differences in the mass density are visualized by color changes representing mass increases of 500 D/1.00 nm 2 . As depicted, binding of xl-mtTFA tetramers to the promoter activation site in linear DNA induced a sharp bend in the DNA duplex in most ( A , B and C ), but not all ( D ) complexes. Arrows mark proximal ends of DNA fragments. The white bar in (A) represents 10 nm.
Source publication
The mitochondrial HMG-box transcription factor xl-mtTFA activates bidirectional transcription by binding to a site separating two core promoters in Xenopus laevis mitochondrial DNA (mtDNA). Three independent approaches were used to study the higher order structure of xl-mtTFA binding to this site. First, co-immunoprecipitation of differentially tag...
Context in source publication
Citations
... It demonstrates anomalous sedimentation behaviour, whereby its self-association ability does not appear to be affected by the presence of DNA. The protein exists in complexes as large as tetramers in the absence of DNA, and it binds the promoter activation site as tetramers [59]. This suggested that the self-association of HMGB1 can also happen in the absence of DNA, under specific conditions. ...
HMGB1, a chromatin protein, interacts with DNA and controls gene expression. However, when HMGB1 is released from apoptotic or damaged cells it triggers pro-inflammatory reactions by interacting with various receptors, mainly RAGE and TLRs. The self-association of HMGB1 has been found to be crucial for its DNA-related biological functions. It is influenced by several factors, such as ionic strength, pH, specific divalent metal cations, redox environment and acetylation. This self-association may also play a role in the interaction with RAGE and TLRs and the concomitant inflammatory responses. Future studies should address the potential role of HMGB1 self-association on its interactions with DNA, RAGE and TLRs, as well as the influence of physico-chemical factors in different cellular environments on these interactions. This article is protected by copyright. All rights reserved.
... Biochemical and electron microscope analyses have shown that xl-mtTFA, a HMG box containing protein of Xenopus laevis, binds DNA as a tetramer in vitro (Antoshechkin et al. 1997). More recent studies showed that the human ortholog of xl-mtTFA (h-mtTFA/TFAM) exists as a monomer or dimer in the absence or presence of DNA, respectively (Kaufman et al. 2007;Gangelhoff et al. 2009). ...
Hmo1, a member of HMGB family proteins in Saccharomyces cerevisiae, binds to and regulates the transcription of genes encoding ribosomal RNA and ribosomal proteins. The functional motifs of Hmo1 include two HMG-like motifs, box A and box B, and a C-terminal tail. To elucidate the molecular roles of the HMG-like boxes in DNA binding in vivo, we analyzed the DNA-binding activity of various Hmo1 mutants using ChIP or reporter assays that enabled us to conveniently detect Hmo1 binding to the promoter of RPS5, a major target gene of Hmo1. Our mutational analyses showed that box B is a bona fide DNA-binding motif and that it also plays other important roles in cell growth. However, box A, especially its first α-helix, contributes to DNA binding of Hmo1 by inducing self-assembly of Hmo1. Intriguingly, box A mediated formation of oligomers of more than two proteins on DNA in vivo. Furthermore, duplication of the box B partially alleviates the requirement for box A. These findings suggest that the principal role of box A is to assemble multiple box B in the appropriate orientation, thereby stabilizing the binding of Hmo1 to DNA and nucleating specific chromosomal architecture on its target genes.
... This characteristic appears to be conserved from Caenorhabditis elegans to humans because HMG-5, a TFAM homologue of C. elegans, is also known to interact with itself in vitro . Xenopus TFAM was previously reported to self-associate and form oligomers in vitro (Antoshechkin et al. 1997). Although several groups have identified the interaction domain of human TFAM in vitro (Gangelhoff et al. 2009;Wong et al. 2009;Ngo et al. 2014), the genuine interaction domain has not yet been determined, and its interpretation is controversial. ...
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 the first time, visualized the interaction of TFAM molecules in mitochondria and proposed its implications for the dynamics of mtDNA nucleoids.
... A cell-free mRNA decay system (41,59) was employed to assess the stability of mitochondrial transcription factor A (Tfam), peroxisome proliferator-activated receptor gamma coactivator 1␣ (PGC-1␣), and nuclear respiratory factor 2␣ (NRF-2␣) because of their critical functions in the process of organelle biogenesis. Tfam contributes to the synthesis of mitochondrially encoded proteins via its participation in the transcription, replication, and maintenance of mitochondrial DNA (mtDNA) (1,19). DNA-binding NRF-2␣ works in conjunction with the coactivator PGC-1␣ to upregulate the expression of several mitochondrial-related genes, including Tfam (62). ...
A change in mRNA stability alters the abundance of mRNA available for translation and is emerging as a critical pathway influencing gene expression. Variations in the stability of functional and regulatory mitochondrial proteins may contribute to the divergent mitochondrial densities observed in striated muscle. Thus we hypothesized that the stability of mRNAs encoding for regulatory nuclear and mitochondrial transcription factors would be inversely proportional to muscle oxidative capacity and would be facilitated by the activity of RNA binding proteins (RBPs). The stability of mitochondrial transcription factor A (Tfam), peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), and nuclear respiratory factor 2α (NRF-2α) mRNA was assessed in striated muscles with distinct oxidative capacities using in vitro decay assays. All three mitochondrial regulators were rapidly degraded in cardiac and slow-twitch red (STR) muscle, resulting in a ∼60-65% lower (P < 0.05) mRNA half-life (t(1/2)) compared with fast-twitch white (FTW) fibers. This accelerated rate of Tfam mRNA decay was matched by a 2.5-fold increase in Tfam transcription in slow- compared with fast-twitch muscle (P = 0.05). Protein expression of four unique RBPs [AU-rich binding factor 1 (AUF1), human antigen R (HuR), KH-homology splicing regulatory protein (KSRP), and CUG binding protein 1 (CUGBP1)] believed to modulate mRNA stability was elevated in cardiac and STR muscles (P < 0.05) and was moderately associated with the decay of Tfam, PGC-1α, and NRF-2α mRNA. Variable rates of transcript degradation were apparent when comparing all transcripts within the same muscle type. Thus the distribution of RBPs appears to follow a fiber-type specific pattern and subsequently functions to alter the stability of specific mitochondrial regulators in a transcript- and tissue-specific fashion.
... mtTFA structure regulates the association with TRX2. It has been shown that mtTFA formed multimers under physiological conditions (27). To confirm whether the interaction of TRX2 with mtTFA requires a specific structure of mtTFA, we introduced mutations at two cysteine residues, positions 49 and 246, as shown in Fig. 4A. ...
Mitochondrial transcription factor A (mtTFA) is a member of the HMG (high mobility group)-box protein family. We previously showed that mtTFA preferentially binds to both cisplatin-damaged and oxidatively damaged DNA. In this study, we found that expression levels of both mtTFA and the mitochondrial antioxidant protein thioredoxin2 (TRX2) are upregulated in cisplatin-resistant cell lines. In addition, TRX2 directly interacts with mtTFA and enhances its damaged DNA binding activity. The interaction between mtTFA and TRX2 requires the HMG box 1 motif of mtTFA. Furthermore, when amino acid substitutions were introduced at either C49G or C246stop, TRX2 interacted with mtTFA. However, the interaction of TRX2 with mtTFA was enhanced when both mutations (C49G and C246stop) were introduced. Binding to cisplatin-damaged DNA was similar among mutant mtTFA proteins. By contrast, binding to oxidized DNA was significantly enhanced when double mutations were introduced. These results suggest that TRX2 not only functions as an antioxidant, but also supports mtTFA functions.
... In HeLa cells, 900-1700 molecules of TFAM were reported to be associated with one molecule of mtDNA (2). It has been estimated that TFAM occupies $25 bp, therefore 900 molecules of TFAM would be enough to coat the entire mitochondrial genome (11)(12)(13). Accordingly, atomic force microscopy experiments have shown that TFAM alone has the capacity to fully compact mtDNA (14). However, it is still not known whether mtDNA is fully saturated with TFAM in vivo. ...
To characterize the organization of mtDNA–protein complexes (known as nucleoids) in vivo, we have probed the mtDNA surface exposure using site-specific DNA methyltransferases targeted to the mitochondria. We have
observed that DNA methyltransferases have different accessibility to different sites on the mtDNA based on the levels of protein
occupancy. We focused our studies on selected regions of mtDNA that are believed to be major regulatory regions involved in
transcription and replication. The transcription termination region (TERM) within the tRNALeu(UUR) gene was consistently and strongly protected from methylation, suggesting frequent and high affinity binding of mitochondrial
transcription termination factor 1 (mTERF1) to the site. Protection from methylation was also observed in other regions of
the mtDNA, including the light and heavy strand promoters (LSP, HSP) and the origin of replication of the light strand (OL).
Manipulations aiming at increasing or decreasing the levels of the mitochondrial transcription factor A (TFAM) led to decreased
in vivo methylation, whereas manipulations that stimulated mtDNA replication led to increased methylation. We also analyzed the effect
of ATAD3 and oxidative stress in mtDNA exposure. Our data provide a map of human mtDNA accessibility and demonstrate that
nucleoids are dynamically associated with proteins.
... The binding site consists of a pair of palindromic sequences that are located between positions 59 (there is one nucleotide overlap with one of the TAS containing repeats) and 91. The consensus for the 2 Xenoturbella binding sites can be seen in figure 2C and is almost identical to the consensus found in Xenopus [46]. It is also possible to identify another pair of putative binding sites at position 130 which partially overlap, rather than being separated by a 0-7 nucleotide gap, as expected in a binding site for multimeric DNA binding factors. ...
Background
Mitochondrial genome comparisons contribute in multiple ways when inferring animal relationships. As well as primary sequence data, rare genomic changes such as gene order, shared gene boundaries and genetic code changes, which are unlikely to have arisen through convergent evolution, are useful tools in resolving deep phylogenies. Xenoturbella bocki is a morphologically simple benthic marine worm recently found to belong among the deuterostomes. Here we present analyses comparing the Xenoturbella bocki mitochondrial gene order, genetic code and control region to those of other metazoan groups.
Results
The complete mitochondrial genome sequence of Xenoturbella bocki was determined. The gene order is most similar to that of the chordates and the hemichordates, indicating that this conserved mitochondrial gene order might be ancestral to the deuterostome clade. Using data from all phyla of deuterostomes, we infer the ancestral mitochondrial gene order for this clade. Using inversion and breakpoint analyses of metazoan mitochondrial genomes, we test conflicting hypotheses for the phylogenetic placement of Xenoturbella and find a closer affinity to the hemichordates than to other metazoan groups. Comparative analyses of the control region reveal similarities in the transcription initiation and termination sites and origin of replication of Xenoturbella with those of the vertebrates. Phylogenetic analyses of the mitochondrial sequence indicate a weakly supported placement as a basal deuterostome, a result that may be the effect of compositional bias.
Conclusion
The mitochondrial genome of Xenoturbella bocki has a very conserved gene arrangement in the deuterostome group, strikingly similar to that of the hemichordates and the chordates, and thus to the ancestral deuterostome gene order. Similarity to the hemichordates in particular is suggested by inversion and breakpoint analysis. Finally, while phylogenetic analyses of the mitochondrial sequences support a basal deuterostome placement, support for this decreases with the use of more sophisticated models of sequence evolution.
... In these later stages, we frequently observe multiple DNA molecules coordinated and linked at a single nexus. Loop structures were not described in AFM studies of Abf2p (Friddle et al., 2004) or in EM analysis of xl-TFAM (Antoshechkin et al., 1997). ...
Packaging DNA into condensed structures is integral to the transmission of genomes. The mammalian mitochondrial genome (mtDNA) is a high copy, maternally inherited genome in which mutations cause a variety of multisystem disorders. In all eukaryotic cells, multiple mtDNAs are packaged with protein into spheroid bodies called nucleoids, which are the fundamental units of mtDNA segregation. The mechanism of nucleoid formation, however, remains unknown. Here, we show that the mitochondrial transcription factor TFAM, an abundant and highly conserved High Mobility Group box protein, binds DNA cooperatively with nanomolar affinity as a homodimer and that it is capable of coordinating and fully compacting several DNA molecules together to form spheroid structures. We use noncontact atomic force microscopy, which achieves near cryo-electron microscope resolution, to reveal the structural details of protein-DNA compaction intermediates. The formation of these complexes involves the bending of the DNA backbone, and DNA loop formation, followed by the filling in of proximal available DNA sites until the DNA is compacted. These results indicate that TFAM alone is sufficient to organize mitochondrial chromatin and provide a mechanism for nucleoid formation.
... However, ratios of X. laevis mtTFA (xl-mtTFA):mtDNA are greatly up-regulated during Xenopus oocyte maturation ranging from a resting immature oocyte level of ∼200:1 (35) to the noted ratio of ∼2000:1 (9), which occurs only in mature oocytes. Given that xl-mtTFA binds mtDNA as a tetramer (36), there is effectively ∼50 xl-mtTFA complexes per genome in immature oocytes, which we argue is a cell type that is more relevant for comparison to mammalian cell types than a mature oocyte, which has dramatically up-regulated mitochondria and mtDNA in preparation for fertilization and development. Finally, if h-mtTFA levels were indeed high enough to completely coat the mtDNA genome as suggested by Kang and colleagues (7,31,33,34), this would seem incompatible with any significant transcriptional output based on in vitro transcription studies (2,37). ...
Human mitochondrial transcription requires the bacteriophage-related RNA polymerase, POLRMT, the mtDNA-binding protein, h-mtTFA/TFAM,
and two transcription factors/rRNA methyltransferases, h-mtTFB1 and h-mtTFB2. Here, we determined the steady-state levels
of these core transcription components and examined the consequences of purposeful elevation of h-mtTFB1 or h-mtTFB2 in HeLa
cells. On a per molecule basis, we find an ∼6-fold excess of POLRMT to mtDNA and ∼3-fold more h-mtTFB2 than h-mtTFB1. We also estimate h-mtTFA at ∼50 molecules/mtDNA, a ratio predicted to support robust transcription, but not to coat mtDNA. Consistent with a role for h-mtTFB2
in transcription and transcription-primed replication, increased mitochondrial DNA and transcripts result from its over-expression.
This is accompanied by increased translation rates of most, but not all mtDNA-encoded proteins. Over-expression of h-mtTFB1
did not significantly influence these parameters, but did result in increased mitochondrial biogenesis. Furthermore, h-mtTFB1
mRNA and protein are elevated in response to h-mtTFB2 over-expression, suggesting the existence of a retrograde signal to
the nucleus to coordinately regulate expression of these related factors. Altogether, our results provide a framework for
understanding the regulation of human mitochondrial transcription in vivo and define distinct roles for h-mtTFB1 and h-mtTFB2 in mitochondrial biogenesis and gene expression that together likely
fine-tune mitochondrial function.
... The core packaging elements of mt-nucleoids are mainly non-histone, HIGH MOBILITY GROUP HMG PROTEINS that show homology to the DNA-binding HMG proteins of nuclear chromatin 15 . These positively charged mtDNA-binding proteins contain two HMG boxes and are evolutionarily conserved from yeast to humans [16][17][18][19][20][21][22][23][24][25][26] . The best-studied HMG protein in mt-nucleoids is yeast Abf2. ...
Mitochondrial DNA (mtDNA) encodes essential components of the cellular energy-producing apparatus, and lesions in mtDNA and mitochondrial dysfunction contribute to numerous human diseases. Understanding mtDNA organization and inheritance is therefore an important goal. Recent studies have revealed that mitochondria use diverse metabolic enzymes to organize and protect mtDNA, drive the segregation of the organellar genome, and couple the inheritance of mtDNA with cellular metabolism. In addition, components of a membrane-associated mtDNA segregation apparatus that might link mtDNA transmission to mitochondrial movements are beginning to be identified. These findings provide new insights into the mechanisms of mtDNA maintenance and inheritance.
