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Background: Recent findings enlightened the pivotal role of cyclooxygenases-1 and -2 (COX-1 and COX-2) in human diseases with inflammation as the committed earliest stage, such as cancer and neurodegenerative diseases. COXs are the main targets of nonsteroidal anti-inflammatory drugs and catalyze the bis-oxygenation of arachidonic acid into prostaglandin PGH2, then converted into prostaglandins, thromboxane, and prostacyclin by tissue-specific isomerases. A remarkable amount of pure COX-1 is necessary to investigate COX-1 structure and function, as well as for in vitro disease biochemical pathway investigations. Methods: Spodoptera frugiperda cells were infected with Baculovirus that revealed to be an efficient expression system to obtain a high amount of ovine(o)COX-1. Protein solubilization time in the presence of a non-ionic detergent was modified, and a second purification step was introduced. Results and Discussion: An improvement of a previously reported method for pure recombinant oCOX-1 production and isolation has been achieved, leading to a lower starting volume of infected cells for each purification, an increased cell density, an increased number of viral particles per cell, and a shortened infection period. The protocol for the recombinant oCOX-1 expression and purification has been in-depth elaborated to obtain 1 mg/L of protein. Conclusion: The optimized procedure could be suitable for producing other membrane proteins as well, for which an improvement in the solubilization step is necessary to have the availability of high concentration proteins.

The m.3243A&gt, G mutation within the mitochondrial mt-tRNALeu(UUR) gene is the most prevalent variant linked to mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS) syndrome. This pathogenic mutation causes severe impairment of mitochondrial protein synthesis due to alterations of the mutated tRNA, such as reduced aminoacylation and a lack of post-transcriptional modification. In transmitochondrial cybrids, overexpression of human mitochondrial leucyl-tRNA synthetase (LARS2) has proven effective in rescuing the phenotype associated with m.3243A&gt, G substitution. The rescuing activity resides in the carboxy-terminal domain (Cterm) of the enzyme, however, the precise molecular mechanisms underlying this process have not been fully elucidated. To deepen our knowledge on the rescuing mechanisms, we demonstrated the interactions of the Cterm with mutated mt-tRNALeu(UUR) and its precursor in MELAS cybrids. Further, the effect of Cterm expression on mitochondrial functions was evaluated. We found that Cterm ameliorates de novo mitochondrial protein synthesis, whilst it has no effect on mt-tRNALeu(UUR) steady-state levels and aminoacylation. Despite the complete recovery of cell viability and the increase in mitochondrial translation, Cterm-overexpressing cybrids were not able to recover bioenergetic competence. These data suggest that, in our MELAS cell model, the beneficial effect of Cterm may be mediated by factors that are independent of the mitochondrial bioenergetics.

ADCA-DN and HSN-IE are rare neurodegenerative syndromes caused by dominant mutations in the replication foci targeting sequence (RFTS) of the DNA methyltransferase 1 (DNMT1) gene. Both phenotypes resemble mitochondrial disorders, and mitochondrial dysfunction was first observed in ADCA-DN. To explore mitochondrial involvement, we studied the effects of DNMT1 mutations in fibroblasts from four ADCA-DN and two HSN-IE patients. We documented impaired activity of purified DNMT1 mutant proteins, which in fibroblasts results in increased DNMT1 amount. We demonstrated that DNMT1 is not localized within mitochondria, but it is associated with the mitochondrial outer membrane. Concordantly, mitochondrial DNA failed to show meaningful CpG methylation. Strikingly, we found activated mitobiogenesis and OXPHOS with significant increase of H2O2, sharply contrasting with a reduced ATP content. Metabolomics profiling of mutant cells highlighted purine, arginine/urea cycle and glutamate metabolisms as the most consistently altered pathways, similar to primary mitochondrial diseases. The most severe mutations showed activation of energy shortage AMPK-dependent sensing, leading to mTORC1 inhibition. We propose that DNMT1 RFTS mutations deregulate metabolism lowering ATP levels, as a result of increased purine catabolism and urea cycle pathways. This is associated with a paradoxical mitochondrial hyper-function and increased oxidative stress, possibly resulting in neurodegeneration in non-dividing cells.

The respiratory complexes are organized in supramolecular assemblies called supercomplexes thought to optimize cellular metabolism under physiological and pathological conditions. In this study, we used genetically and biochemically well characterized cells bearing the pathogenic microdeletion m.15,649–15,666 (ΔI300-P305) in MT-CYB gene, to investigate the effects of an assembly-hampered CIII on the re-organization of supercomplexes. First, we found that this mutation also affects the stability of both CI and CIV, and evidences the occurrence of a preferential structural interaction between CI and CIII(2), yielding a small amount of active CI+CIII(2) super-complex. Indeed, a residual CI+CIII combined redox activity, and a low but detectable ATP synthesis driven by CI substrates are detectable, suggesting that the assembly of CIII into the CI+CIII(2) supercomplex mitigates the detrimental effects of MT-CYB deletion. Second, measurements of oxygen consumption and ATP synthesis driven by NADH-linked and FADH(2)-linked substrates alone, or in combination, indicate a common ubiquinone pool for the two respiratory pathways. Finally, we report that prolonged incubation with rotenone enhances the amount of CI and CIII(2), but reduces CIV assembly. Conversely, the antioxidant N-acetylcysteine increases CIII(2) and CIV(2) and partially restores respirasome formation. Accordingly, after NAC treatment, the rate of ATP synthesis increases by two-fold compared with untreated cell, while the succinate level, which is enhanced by the homoplasmic mutation, markedly decreases. Overall, our findings show that fine-tuning the supercomplexes stability improves the energetic efficiency of cells with the MT-CYB microdeletion.

Replication of mammalian mitochondrial DNA (mtDNA) is an essential process that requires high fidelity and control at multiple levels to ensure proper mitochondrial function. Mutations in the mitochondrial genome maintenance exonuclease 1 (MGME1) gene were recently reported in mitochondrial disease patients. Here, to study disease pathophysiology, we generated Mgme1 knockout mice and report that homozygous knockouts develop depletion and multiple deletions of mtDNA. The mtDNA replication stalling phenotypes vary dramatically in different tissues of Mgme1 knockout mice. Mice with MGME1 deficiency accumulate a long linear subgenomic mtDNA species, similar to the one found in mtDNA mutator mice, but do not develop progeria. This finding resolves a long-standing debate by showing that point mutations of mtDNA are the main cause of progeria in mtDNA mutator mice. We also propose a role for MGME1 in the regulation of replication and transcription termination at the end of the control region of mtDNA., It has been debated whether premature ageing in mitochondrial DNA mutator mice is driven by point mutations or deletions of mtDNA. Matic et al generate Mgme1 knockout mice and show here that these mice have tissue-specific replication stalling and accumulate deleted mtDNA, without developing progeria.

Mammalian mitochondrial DNA (mtDNA) is packaged by mitochondrial transcription factor A (TFAM) into mitochondrial nucleoids that are of key importance in controlling the transmission and expression of mtDNA. Nucleoid ultrastructure is poorly defined, and therefore we used a combination of biochemistry, superresolution microscopy, and electron microscopy to show that mitochondrial nucleoids have an irregular ellipsoidal shape and typically contain a single copy of mtDNA. Rotary shadowing electron microscopy revealed that nucleoid formation in vitro is a multistep process initiated by TFAM aggregation and cross-strand binding. Superresolution microscopy of cultivated cells showed that increased mtDNA copy number increases nucleoid numbers without altering their sizes. Electron cryo-tomography visualized nucleoids at high resolution in isolated mammalian mitochondria and confirmed the sizes observed by superresolution microscopy of cell lines. We conclude that the fundamental organizational unit of the mitochondrial nucleoid is a single copy of mtDNA compacted by TFAM, and we suggest a packaging mechanism.

Mitochondrial transcription factor A (TFAM) is a key component for the protection and transcription of the mitochondrial genome. TFAM belongs to the high mobility group (HMG) box family of DNA binding proteins that are able to bind to and bend DNA. Human TFAM (huTFAM) contains two HMG box domains separated by a linker region, and a 26 amino acid C-terminal tail distal to the second HMG box. Previous studies on huTFAM have shown that requisites for proper DNA bending and specific binding to the mitochondrial genome are specific intercalating residues and the C-terminal tail. We have characterized TFAM from the sea urchin Paracentrotus lividus (suTFAM). Differently from human, suTFAM contains a short 9 amino acid C-terminal tail, yet it still has the ability to specifically bind to mtDNA. To provide information on the mode of binding of the protein we used fluorescence resonance energy transfer (FRET) assays and found that, in spite of the absence of a canonical C-terminal tail, suTFAM distorts DNA at a great extent and recognizes specific target with high affinity. Site directed mutagenesis showed that the two Phe residues placed in corresponding position of the two intercalating Leu of huTFAM are responsible for the strong bending and the great binding affinity of suTFAM.

Sir, Leber’s hereditary optic neuropathy (LHON) is one of the best-studied mitochondrial diseases (for review see Carelli et al. , 2004; Yu-Wai-Mann et al. , 2011). Classically, there is a subacute loss of central vision that affects prevalently males, peaking in young adulthood. After a few months, optic atrophy becomes evident and the patient enters a chronic phase, in most cases characterized by stable blindness. Infrequently a partial recovery of visual acuity occurs. Exceptions have been reported. Concerning age of onset, childhood cases seem to represent a different version of LHON that often resembles dominant optic atrophy (Barboni et al. , 2006), whereas very late-onset cases have also been reported, widening the spectrum (Shah et al. , 2008; Yu-Wai-Man et al. , 2008; Giraudet et al. , 2011; Dimitriadis et al. , 2014). There is also a certain degree of heterogeneity in clinical expression, ranging from mild to severe cases, and the rates of spontaneous recovery of vision vary with mutation type (Carelli et al. , 2004, 2011; Yu-Wai-Mann et al. , 2011). A maternally inherited mitochondrial DNA (mtDNA) mutation is necessary but not sufficient to develop optic neuropathy. There are three common mtDNA mutations affecting complex I (i.e. m.11778G>A/MT-ND4, m.3460G>A/MT-ND1 and m.14484T>C/MT-ND6). Most cases remain as asymptomatic carriers, with males much more likely than females to undergo conversion to the affected state. Thus, male prevalence and incomplete penetrance remain areas of active investigation. Environmental factors have now been demonstrated as frequent triggers of the disease. The most common is tobacco smoke, in combination with or without alcohol consumption (Sadun et al. , 2003), as corroborated by Kirkman and colleagues (2009) with their large survey of LHON affected and unaffected mutation carriers. More rarely, exposure to toxins, various forms of smoke, or certain …

Mitochondrial transcription for replication primer formation has priority over gene expression at low POLRMT levels., Mitochondria are vital in providing cellular energy via their oxidative phosphorylation system, which requires the coordinated expression of genes encoded by both the nuclear and mitochondrial genomes (mtDNA). Transcription of the circular mammalian mtDNA depends on a single mitochondrial RNA polymerase (POLRMT). Although the transcription initiation process is well understood, it is debated whether POLRMT also serves as the primase for the initiation of mtDNA replication. In the nucleus, the RNA polymerases needed for gene expression have no such role. Conditional knockout of Polrmt in the heart results in severe mitochondrial dysfunction causing dilated cardiomyopathy in young mice. We further studied the molecular consequences of different expression levels of POLRMT and found that POLRMT is essential for primer synthesis to initiate mtDNA replication in vivo. Furthermore, transcription initiation for primer formation has priority over gene expression. Surprisingly, mitochondrial transcription factor A (TFAM) exists in an mtDNA-free pool in the Polrmt knockout mice. TFAM levels remain unchanged despite strong mtDNA depletion, and TFAM is thus protected from degradation of the AAA+ Lon protease in the absence of POLRMT. Last, we report that mitochondrial transcription elongation factor may compensate for a partial depletion of POLRMT in heterozygous Polrmt knockout mice, indicating a direct regulatory role of this factor in transcription. In conclusion, we present in vivo evidence that POLRMT has a key regulatory role in the replication of mammalian mtDNA and is part of a transcriptional mechanism that provides a switch between primer formation for mtDNA replication and mitochondrial gene expression.

The MTERF family is a large protein family, identified in metazoans and plants, which consists of four subfamilies, MTERF1, 2, 3 and 4. Mitochondrial localisation was predicted for the vast majority of MTERF family members and demonstrated for the characterised MTERF proteins. The main structural feature of MTERF proteins is the presence of a modular architecture, based on repetitions of a 30-residue module, the mTERF motif, containing leucine zipper-like heptads. The MTERF family includes transcription termination factors: human mTERF, sea urchin mtDBP andDrosophilaDmTTF. In addition to terminating transcription, they are involved in transcription initiation and in the control of mtDNA replication. This multiplicity of functions seems to flank differences in the gene organisation of mitochondrial genomes. MTERF2 and MTERF3 play antithetical roles in controlling mitochondrial transcription: that is, mammalian andDrosophilaMTERF3 act as negative regulators, whereas mammalian MTERF2 functions as a positive regulator. Both proteins contact mtDNA in the promoter region, perhaps establishing interactions, either mutual or with other factors. Regulation of MTERF gene expression in human andDrosophiladepends on nuclear transcription factors NRF-2 and DREF, respectively, and proceeds through pathways which appear to discriminate between factors positively or negatively acting in mitochondrial transcription. In this emerging scenario, it appears that MTERF proteins act to coordinate mitochondrial transcription.

In mammals, NRF-2 (nuclear respiratory factor 2), also named GA-binding protein, is an Ets family transcription factor that controls many genes involved in cell cycle progression and protein synthesis as well as in mitochondrial biogenesis. In this paper, we analyzed the role of NRF-2 in the regulation of human genes involved in mitochondrial DNA transcription and replication. By a combination of bioinformatic and biochemical approaches, we found that the factor binds in vitro and in vivo to the proximal promoter region of the genes coding for the transcription termination factor mTERF, the RNA polymerase POLRMT, the B subunit of the DNA polymerase-gamma, the DNA helicase TWINKLE, and the single-stranded DNA-binding protein mtSSB. The role of NRF-2 in modulating the expression of those genes was further established by RNA interference and overexpression strategies. On the contrary, we found that NRF-2 does not control the genes for the subunit A of DNA polymerase-gamma and for the transcription repressor MTERF3; we suggest that these genes are under regulatory mechanisms that do not involve NRF proteins. Since NRFs are known to positively control the expression of transcription-activating proteins, the novelty emerging from our data is that proteins playing antithetical roles in mitochondrial DNA transcription, namely activators and repressors, are under different regulatory pathways. Finally, we developed a more stringent consensus with respect to the general consensus of NRF-2/GA-binding protein when searching for NRF-2 binding sites in the promoter of mitochondrial proteins.

The MTERF-family is a wide family of proteins identified in Metazoa and plants which includes the known mitochondrial transcription termination factors. With the aim to shed light on the function of MTERF-family members in Drosophila, we performed the cloning and characterization of D-MTERF3, a component of the most conserved group of this family. D-MTERF3 is a mitochondrial protein of 323 amino acids. Sequence analysis in seven different organisms showed that the protein contains five conserved “mTERF-motifs”, three of which include a leucine zipper-like domain. D-MTERF3 knock-down, obtained by RNAi in D.Mel-2 cells, did not affect mitochondrial replication and transcription. On the contrary, it decreased to a variable extent the rate of labelling of about half of the mitochondrial polypeptides, with ND1 being the most affected by D-MTERF3 depletion. These results indicate that D-MTERF3 is involved in mitochondrial translation. This role, likely based on protein–protein interactions, may be exerted either through a direct interaction with the translation machinery or by bridging the mitochondrial transcription and translation apparatus.

DmTTF is a Drosophila mitochondrial DNA-binding protein, which recognizes two sequences placed at the boundary of clusters of genes transcribed in opposite directions. To obtain in vivo evidences on the role of DmTTF, we characterized a DmTTF knock-down phenotype obtained by means of RNA interference in D.Mel-2 cells. By a combination of RNase protection and real-time RT-PCR experiments we found that knock-down determines remarkable changes in mitochondrial transcription. In particular, protein depletion increases not only the level of (+) and (-)strand RNAs mapping immediately after of the two protein-binding site, but also that of transcripts located further downstream. Unexpectedly, depletion of the protein also causes the decrease in the content of those transcripts mapping upstream of the protein target sites, including the two rRNAs. The changes in transcript level do not depend on a variation in mitochondrial DNA (mtDNA) content, since mtDNA copy number is unaffected by DmTTF depletion. This work shows conclusively that DmTTF arrests in vivo the progression of the mitochondrial RNA polymerase; this is the first ever-obtained evidence for an in vivo role of an animal mitochondrial transcription termination factor. In addition, the reported data provide interesting insights into the involvement of DmTTF in transcription initiation in Drosophila mitochondria.

DmTTF is a Drosophila melanogaster mitochondrial DNA-binding protein which binds specifically to two homologous non-coding sequences located at the 30 ends of blocks of genes encoded on opposite strands. In order to test whether this protein acts as transcription termination factor, we assayed the capacity of DmTTF to arrest in vitro the transcription catalyzed by mitochondrial and bacteriophage RNA polymerases. Experiments with human S-100 extracts showed that DmTTF is able to arrest the transcription catalyzed by human mitochondrial RNA polymerase bidirectionally, independently of the orientation of the protein–DNA complex. On the contrary when T3 or T7 RNA polymerases were used, we found that DmTTF prevalently arrests transcription when the DNA-binding site was placed in the reverse orientation with respect to the incoming enzymes. These results demonstrate that DmTTF is a transcription termination factor with a biased polarity and suggest that the DNA-bound protein is structurally asymmetrical, exposing two different faces to RNA polymerases travelling on opposite directions.

The glutathione S-transferase (GST) fusion protein system is widely used for high-level expression and efficient purification of recombinant proteins from bacteria. However many GST-tagged proteins are insoluble, and the existing procedures, which employ a mixture of detergents to solubilize the molecules, frequently compromise their functional activity. A further limitation is that large proteins (>80 kDa) are poorly isolated by the current methods and are contaminated by truncated forms. To overcome these problems, we provide here an improved method for efficient purification of active large GST-tagged enzymes such as the 180-kDa GST-fused mitochondrial RNA polymerase. (C) 2011 Elsevier Inc. All rights reserved.

GST fusion proteins expressed in bacteria often tend to form aggregates and are inefficiently purified by standard procedures, which employ a mixture of detergents that compromise the binding efficiency to the affinity resin and the biological activity of the recombinant proteins. Moreover, the binding to the resin is negatively affected by the molecular weight of the fusion protein. Here we report a simple and efficient method to purify active large GST-tagged proteins, which uses high ionic strength buffer to solubilize the protein aggregates in a bacterial lysate. Affinity-chromatography purification is achieved by adopting two columns connected in series, which facilitate the binding of large GST fused molecules. This approach was applied to purify the 180-kDa GST-tagged mitochondrial RNA polymerase. We also report conditions for simple and efficient GST tag removal from the eluted protein. Finally we demonstrate that the recombinant enzyme is capable to catalyze RNA synthesis.

Sea urchin mtDNA is transcribed via a different mechanism compared to vertebrates. To gain information on the apparatus of sea urchin mitochondrial transcription we have characterized the DNA binding properties of the mitochondrial transcription factor A (TFAM). The protein contains two HMG box domains but, differently from vertebrates, displays a very short C-terminal tail. Phylogenetic analysis showed that the distribution of tail length is mixed in the different lineages, indicating that it is a trait that undergoes rapid changes during evolution. Homology modeling suggests that the protein adopts the same configuration of the human counterpart and possibly a similar mode of binding to DNA. DNase I footprinting showed that TFAM specifically contacts mtDNA at a fixed distance from three AT-rich consensus sequences that were supposed to act as transcriptional initiation sites. Bound sequences are homologous and, contain an inverted repeat motif, which resembles that involved in the intercalation of human TFAM in LSP DNA. The here reported data indicate that sea urchin TFAM specifically binds mtDNA. The protein could intercalate residues at the DNA inverted motif and, despite its short tail, might have a role in mitochondrial transcription. (C) 2013 The Authors. Elsevier B.V. and Mitochondria Research Society. All rights reserved.

GST fusion proteins expressed in bacteria often tend to form aggregates and are inefficiently purified by standard procedures, which employ a mixture of detergents that compromise the binding efficiency to the affinity resin and the biological activity of the recombinant proteins. Moreover, the binding to the resin is negatively affected by the molecular weight of the fusion protein. Here we report a simple and efficient method to purify active large GST-tagged proteins, which uses high ionic strength buffer to solubilize the protein aggregates in a bacterial lysate. Affinity-chromatography purification is achieved by adopting two columns connected in series, which facilitate the binding of large GST fused molecules. This approach was applied to purify the 180-kDa GST-tagged mitochondrial RNA polymerase. We also report conditions for simple and efficient GST tag removal from the eluted protein. Finally we demonstrate that the recombinant enzyme is capable to catalyze RNA synthesis.

To study the molecular mechanisms responsible for the regulation of mitochondrial DNA copy number, in vivo and in organello dimethyl sulfate footprinting experiments in human fibroblasts and rat liver mitochondria were carried out. By this approach we identified in both species two specific protein binding sites in the 3′ region of the displacement loop of mitochondrial DNA. One site contains the TAS-D element of human and rat mitochondrial DNA; the other covers TAS-C and TAS-B in human, whereas in rat it comprises part of TAS-B. We suggest that the protected sequences might be the site of action of protein factors involved in the premature termination of mitochondrial DNA heavy-strand synthesis.

A binding protein for single-stranded DNA was purified from Paracentrotus lividus egg mitochondria to near homogeneity by chromatography on DEAE-Sephacel and single-stranded-DNA-cellulose. The protein consists of a single polypeptide of about 15 kDa. Glycerol gradient sedimentation analysis suggested that P. lividus mitochondrial single-stranded-DNA-binding protein exists as a homo-oligomer, possibly a tetramer, in solution. The protein shows a stronger preference for poly(dT) with respect to single-stranded M13, poly(dI) and poly(dC). Binding to poly(dA) takes place with much lower affinity. The binding-site size, determined by gel mobility-shift experiments with oligonucleotides of different length, is approximately 45 nucleotides. The binding to single-stranded DNA occurs with low or no cooperativity and is not influenced by ionic strength. The protein has a very high affinity for the DNA: its apparent macroscopic association constant is 2x10(9) M(-1), a value which is the highest among the mitochondrial single-stranded-DNA-binding proteins characterized to date. The lack of cooperativity and the high association constant represent distinctive features of this protein and might be related to the peculiar mechanism of sea urchin mitochondrial DNA replication.

et al., DREF [DRE (DNA replication-related element)-binding factor] controls the transcription of numerous genes in Drosophila, many involved in nuclear DNA (nDNA) replication and cell proliferation, three in mitochondrial DNA (mtDNA) replication and two in mtDNA transcription termination. In this work, we have analysed the involvement of DREF in the expression of the known remaining genes engaged in the minimal mtDNA replication (d-mtDNA helicase) and transcription (the activator d-mtTFB2) machineries and of a gene involved in mitochondrial mRNA translation (d-mtTFB1). We have identified their transcriptional initiation sites and DRE sequences in their promoter regions. Gel-shift and chromatin immunoprecipitation assays demonstrate that DREF interacts in vitro and in vivo with the d-mtDNA helicase and d-mtTFB2, but not with the d-mtTFB1 promoters. Transient transfection assays in Drosophila S2 cells with mutated DRE motifs and truncated promoter regions show that DREF controls the transcription of d-mtDNA helicase and d-mtTFB2, but not that of d-mtTFB1. RNA interference of DREF in S2 cells reinforces these results showing a decrease in the mRNA levels of d-mtDNA helicase and d-mtTFB2 and no changes in those of the d-mtTFB1. These results link the genetic regulation of nuclear DNA replication with the genetic control of mtDNA replication and transcriptional activation in Drosophila. © 2013 Elsevier B.V., This work was supported by grants from the Center for Biomedical Research on Rare Diseases (CIBERER) [INTRA/08/743.2] to R.G., the Instituto de Salud Carlos III (grants [PI 07/0167] and [PI 10/0703] to R.G.); the Comunidad de Madrid (grants [GEN-0269/2006] and [MITOLAB S2010/BMD-2402] to R.G); the Università di Bari, Progetto di Ricerca di Ateneo (to M.R.); and the National Institutes of Health (grant [GM45295] to L.S.K.).

As a step toward the characterization of the main components of mitochondrial DNA replication apparatus in sea urchin, we report the identification of a DNA-helicase activity inParacentrotus lividusmitochondria. The activity was detected in a protein fraction obtained by fractionating on DEAE-Sephacel a lysate of gradient purified mitochondria fromParacentrotus lividuseggs. The mitochondrial helicase unwound, in the presence of ATP and Mg++, a 39-base oligonucleotide annealed to single-stranded M13mp18 (+) DNA. Its direction of movement is 3′ to 5′ with respect to the single stranded portion of the partial duplex DNA substrate. This polarity is similar to that exhibited by the single stranded portion of the partial duplex DNA substrate. This polarity is similar to that exhibited by theEscherichia coli rephelicase and by the helicase from bovine brain mitochondria. These features suggest that the sea urchin mitochondrial helicase could function in enabling the polymerization of the H-strand during mitochondrial DNA replication.

Regulation of mitochondrial DNA (mtDNA) expression is critical for the control of oxidative phosphorylation in response to physiological demand, and this regulation is often impaired in disease and aging. We have previously shown that mitochondrial transcription termination factor 3 (MTERF3) is a key regulator that represses mtDNA transcription in the mouse, but its molecular mode of action has remained elusive. Based on the hypothesis that key regulatory mechanisms for mtDNA expression are conserved in metazoans, we analyzed Mterf3 knockout and knockdown flies. We demonstrate here that decreased expression of MTERF3 not only leads to activation of mtDNA transcription, but also impairs assembly of the large mitochondrial ribosomal subunit. This novel function of MTERF3 in mitochondrial ribosomal biogenesis is conserved in the mouse, thus we identify a novel and unexpected role for MTERF3 in coordinating the crosstalk between transcription and translation for the regulation of mammalian mtDNA gene expression., Author Summary One of the main functions of the mitochondrial network is to provide the energy currency ATP to drive a large array of cellular metabolic processes. The formation of the mitochondrial respiratory chain, which allows this energy supply, is under the control of two separate genetic systems, the nuclear and the mitochondrial genomes, whose expressions have to be tightly coordinated to ensure efficient mitochondrial function. The regulation of mitochondrial genome expression is still poorly understood despite the profound importance of this process in human physiology, disease, and aging. Here, we make one step forward by unraveling a new role for the mitochondrial transcription termination factor 3 (MTERF3), which was initially characterized as a factor able to decrease mitochondrial transcription. Using gene invalidation approaches, we show in two distinct model organisms, the fruit fly and the mouse, that MTERF3 is not only involved in mitochondrial transcription but also favors the assembly of the mitochondrial ribosome and thereby reinforces the coordination between transcription and translation events, two key steps in mitochondrial genome expression.

A mitochondrial protein, able to specifically bind two double-stranded homologous sequences of seaurchin mitochondrial DNA, has been partially purified from Paracentrotus lividus eggs. This protein, present at a low concentration, is a polypeptide of 40 kDa. One of the binding sequences, located in the main non-coding region, contains the replication origin of the mitochondrial DNA H-strand. By a combination of band-shift, DNase footprinting, and modification interference analyses with homologous and heterologous probes we identified YCYYATCAN(A/T)RC as the minimum sequence required for the binding. The protein also shows a single-stranded DNA-binding activity, as it is able to specifically interact with one of the strands of the binding sites. These features are consistent with a function of the protein in the modulation of sea-urchin mitochondrial DNA replication during the developmental stages.

The mechanism of mitochondrial transcription and its apparatus have been investigated in different types of animal cells. While transcription mechanisms are different, due probably to the variation in gene organization among metazoans, the mitochondrial transcription apparatus is substantially conserved. The core mitochondrial transcription machinery is a three-member complex made of RNA polymerase (mtRNAP) and factors TFAM and TFB2M. Other factors, such as the proteins of the mTERF family, participate in this process. The transcription termination factor MTERF1 may also contribute to DNA melting during transcription initiation. MTERF2 and MTERF3 appear to modulate mitochondrial transcription by interacting simultaneously or separately with the core transcription apparatus. Several aspects of mitochondrial transcription need further investigation. These include the mechanisms responsible for the choice of the different initiation sites, the role of auxiliary factors in mitochondrial transcription, and the mechanism of stabilization of mitochondrial RNAs in response to different cell requirements and physiological conditions.

Characterization of the basic transcription machinery of mammalian mitochondrial DNA has been greatly supported by the availability of pure recombinant mitochondrial RNA polymerase (mtRNAP) and accessory factors, which allowed to develop a reconstituted in vitro transcription system. This chapter outlines a general strategy that makes use of a minimal promoter-independent transcription assay to study mitochondrial transcription termination in animal systems. We used such a system to investigate the transcription termination properties of the sea urchin factor mtDBP, however, it is applicable to the study of transcription termination in a variety of organisms, provided that the pure mtRNAP and the transcription termination factor are available.The assay here described contains the recombinant proteins mtRNAP and mtDBP, both expressed in insect cells, and a template consisting of a 3'-tailed DNA construct bearing the sequence bound by mtDBP. Transcription by the RNA polymerase produces run-off and terminated molecules, the size of the latter being consistent with RNA chain arrest in correspondence of the mtDBP-DNA complex. Transcription termination is protein-dependent as addition of increasing amounts of mtDBP to the assay causes a decrease in the intensity of the run-off and the gradual appearance of short-terminated molecules. Furthermore, we report a method, based on pulse-chase experiments, which allows us to distinguish between the true termination and the pausing events.

et al., The DREF [DRE (DNA replication-related element)-binding factor], which regulates the transcription of a group of cell proliferation-related genes in Drosophila, also controls the expression of three genes involved in mtDNA (mitochondrial DNA) replication and maintenance. In the present study, by in silico analysis, we have identified DREs in the promoter region of a gene participating in mtDNA transcription, the DmTTF (Drosophila mitochondrial transcription termination factor). Transient transfection assays in Drosophila S2 cells, with mutated versions of DmTTF promoter region, showed that DREs control DmTTF transcription; moreover, gel-shift and Chip (chromatin immunoprecipitation) assays demonstrated that the analysed DRE sites interact with DREF in vitro and in vivo. Accordingly, DREF knock-down in S2 cells by RNAi (RNA interference) induced it considerable decrease in DmTTF mRNA level. These results clearly demonstrate that DREF positively controls DmTTF expression. On the other hand, mtRNApol (mitochondrial RNA polymerase) lacks DREs in its promoter and is not regulated in vivo by DREF. In situ RNA hybridization Studies showed that DmTTF was transcribed almost ubiquitously throughout all stages of Drosophila embryogenesis, whereas mRNApol was efficiently transcribed from stages 11-12. Territories where transcription occurred mostly were the gut and Malpighi tubes for DmTTF, and the gut, mesoderm, pharyngeal muscle and Malpighi tubes for mtRNApol. The partial overlapping in the temporal and spatial mRNA expression patterns confirms that transcription of the two genes is differentially regulated during embryogenesis and suggests that DmTTF might play multiple roles in the mtDNA transcription process, for which different levels of the protein with respect to mtRNApol are required.

Characterization of the basic transcription machinery of mammalian mitochondrial DNA has been greatly supported by the availability of pure recombinant mitochondrial RNA polymerase (mtRNAP) and accessory factors, which allowed to develop a reconstituted in vitro transcription system. This chapter outlines a general strategy that makes use of a minimal promoter-independent transcription assay to study mitochondrial transcription termination in animal systems. We used such a system to investigate the transcription termination properties of the sea urchin factor mtDBP, however, it is applicable to the study of transcription termination in a variety of organisms, provided that the pure mtRNAP and the transcription termination factor are available.The assay here described contains the recombinant proteins mtRNAP and mtDBP, both expressed in insect cells, and a template consisting of a 3'-tailed DNA construct bearing the sequence bound by mtDBP. Transcription by the RNA polymerase produces run-off and terminated molecules, the size of the latter being consistent with RNA chain arrest in correspondence of the mtDBP-DNA complex. Transcription termination is protein-dependent as addition of increasing amounts of mtDBP to the assay causes a decrease in the intensity of the run-off and the gradual appearance of short-terminated molecules. Furthermore, we report a method, based on pulse-chase experiments, which allows us to distinguish between the true termination and the pausing events.

Termination of transcription is a key process in the regulation of mitochondrial gene expression in animal cells. To investigate transcription termination in sea urchin mitochondria, we cloned the mitochondrial RNA polymerase (mtRNAP) of Paracentrotus lividus and used a recombinant form of the enzyme in a reconstituted transcription system, in the presence of the DNA-binding protein mtDBP. Cloning of mtRNAP was performed by a combination of PCR with degenerate primers and library screening. The enzyme contains 10 phage-like conserved motifs, two pentatricopeptide motifs and a serine-rich stretch. The protein expressed in insect cells supports transcription elongation in a promoter-independent assay. Addition of recombinant mtDBP caused arrest of the transcribing mtRNAP when the enzyme approached the mtDBP-binding site in the direction of transcription of mtDNA l-strand. When the polymerase encountered the protein-binding site in the opposite direction, termination occurred in a protein-independent manner, inside the mtDBP-binding site. Pulse-chase experiments show that mtDBP caused true transcription termination rather than pausing. These data indicate that mtDBP acts as polar termination factor and suggest that transcription termination in sea urchin mitochondria could take place by two alternative modes based on protein-mediated or sequence-dependent mechanisms.

The sea urchin mitochondrial D-loop binding protein (mtDBP) is a transcription termination factor that is able to arrest bidirectionally mitochondrial RNA chain elongation. The observation that the mtDBP binding site in the main non-coding region is located in correspondence of the 3 0 end of the triplex structure, where the synthesis of heavy strand mitochondrial (mt) DNA is either prematurely terminated or allowed to continue, raised the question whether mtDBP could also regulate mtDNA replication. By using a helicase assay in the presence of the replicative helicase of SV40, we show that mtDBP is able to inhibit the enzyme thus acting as a contrahelicase. The impairing activity of mtDBP is bidirectional as it is independent of the orientation of the protein binding site. The inhibition is increased by the presence of the guanosine-rich sequence that flanks mtDBP binding site. Finally, a mechanism of abrogation of mtDBP contrahelicase activity is suggested that is based on the dissociation of mtDBP from DNA caused by the passage of the RNA polymerase through the protein–DNA complex. All these findings favour the view that mtDBP, besides serving as transcription termination factor, could also act as a negative regulator of mtDNA synthesis at the level of D-loop expansion.

Using a combination of bioinformatic and molecular biology approaches a Drosophila melanogaster protein, DmTTF, has been identified, which exhibits sequence and structural similarity with two mitochondrial transcription termination factors, mTERF (human) and mtDBP (sea urchin). Import/processing assays indicate that DmTTF is synthesised as a precursor of 410 amino acids and is imported into mitochondria, giving rise to a mature product of 366 residues. Band-shift and DNase I protection experiments show that DmTTF binds two homologous, short, non-coding sequences of Drosophila mitochondrial DNA, located at the 3' end of blocks of genes transcribed on opposite strands. The location of the target sequences coincides with that of two of the putative transcription termination sites previously hypothesised. These results indicate that DmTTF is the termination factor of mitochondrial transcription in Drosophila. The existence of two DmTTF binding sites might serve not only to stop transcription but also to control the overlapping of a large number of transcripts generated by the peculiar transcription mechanism operating in this organism.

The cloning of the cDNA for two mitochondrial proteins involved in sea urchin mtDNA replication and transcription is reported here. The cDNA for the mitochondrial D-loop binding protein (mtDBP) from the sea urchin Strongylocentrotus purpuratus has been cloned by a polymerase chain reaction-based approach. The protein displays a very high similarity with the Paracentrotus lividus homologue as it contains also the two leucine zipper-like domains which are thought to be involved in intramolecular interactions needed to expose the two DNA binding domains in the correct position for contacting DNA. The cDNA for the mitochondrial single-stranded DNA-binding protein (mtSSB) from P. lividus has been also cloned by a similar approach. The precursor protein is 146 amino acids long with a presequence of 16 residues. The deduced amino acid sequence shows the highest homology with the Xenopus laevis protein and the lowest with the Drosophila mtSSB. The computer modeling of the tertiary structure of P. lividus mtSSB shows a structure very similar to that experimentally determined for human mtSSB, with the conservation of the main residues involved in protein tetramerization and in DNA binding.

The sea urchin mitochondrial displacement (D)-loop binding protein mtDBP has been previously identified and cloned. The polypeptide (348 amino acids) displays a significant homology with the human mitochondrial transcription termination factor mTERF. This similarity, and the observation that the 3' ends of mitochondrial RNAs coded by opposite strands mapped in correspondence of mtDBP-binding sites, suggested that mtDBP could function as transcription termination factor in sea urchin mitochondria. To investigate such a role we tested the capability of mtDBP bound to its target sequence in the main non-coding region to affect RNA elongation by mitochondrial and bacteriophage T3 and T7 RNA polymerases. We show that mtDBP was able to terminate transcription bidirectionally when initiated by human mitochondrial RNA polymerase but only unidirectionally when initiated by T3 or T7 RNA polymerases. Time-course experiments indicated that mtDBP promotes true transcription termination rather than transcription pausing. These results indicate that mtDBP is able to function as a bipolar transcription termination factor in sea urchin mitochondria. The functional significance of such an activity could be linked to the previously proposed dual role of the protein in modulating mitochondrial DNA transcription and replication.

The Paracentrotus lividus mitochondrial D-loop binding protein (mtDBP) is a DNA-binding protein which is involved in the regulation of sea urchin mtDNA transcription. Immunoblots of Heparin Sepharose-bound proteins at selected early developmental stages, as well as electrophoretic mobility shift assay, show that mtDBP is present in the egg at a concentration of about 1 x 10(6) molecules/egg. Its level increases after fertilization of about twofold, remaining substantially unchanged between 16-h blastula stage and early pluteus stage and declines thereafter. The content of mtDBP mRNA, determined by RNase protection experiments, increases about sevenfold at the 16-h blastula stage compared to the egg. A considerable decrease occurs at the 40-h pluteus stage, which precedes that of the protein. These results suggest that the expression of mtDBP is regulated at transcriptional level up to blastula stage, while other factors, in addition to the level of the RNA may control the content of this protein in the following stages of embryogenesis. (C) 2000 Academic Press.

Footprinting studies with the purine-modifying agent dimethyl sulphate were performed in Paracentrotus lividus eggs and embryos to analyze in vivo the interactions between protein and mitochondrial DNA. Footprinting in the small non-coding region and at the boundary between the ND5 and ND6 genes revealed two strong contact sites corresponding with the in vitro binding sequences of mitochondrial D-loop-Binding Protein (mtDBP). The analysis of the pause region of mtDNA replication showed a strong footprint corresponding with the binding site of the mitochondrial Pause region-Binding Protein-2 (mtPBP-2), but only a very weak signal at the binding site of the mitochondrial Pause region-Binding Protein-1 (mtPBP-1), which in vitro binds DNA with high efficiency. In vitro and in vivo analysis of the 3′ end-region of the two rRNA genes showed no significant protein-DNA interactions, suggesting that, in contrast to mammals, the 3′ ends of sea urchin mitochondrial rRNAs are not generated by a protein-dependent transcription termination event. These and other data support a model in which expression of mitochondrial genes in sea urchins is regulated post-transcriptionally. Footprinting at the five AT-rich consensus regions allowed the detection of a binding site in the non-coding region for an as-yet unidentified protein, mtAT-1BP. The occupancy of this site appears to be developmentally regulated, being detectable in the pluteus larval stage, but not in unfertilized eggs.

The cDNA for the sea urchin mitochondrial D-loop-binding protein (mtDBP), a 40 kDa protein which binds two homologous regions of mitochondrial DNA (the D-loop region and the boundary between the oppositely transcribed ND5 and ND6 genes), has been cloned. Four different 3'-untranslated regions have been detected that are related to each other in pairs and do not contain the canonical polyadenylation signal. The in vitro synthesised mature protein (348 amino acids), deprived of the putative signal sequence, binds specifically to its DNA target sequence and produces a DNase I footprint identical to that given by the natural protein. mtDBP contains two leucine zippers, one of which is bipartite, and two small N- and C-terminal basic domains. A deletion mutation analysis of the recombinant protein has shown that the N-terminal region and the two leucine zippers are necessary for the binding. Furthermore, evidence was provided that mtDBP binds DNA as a monomer. This rules out a dimerization role for the leucine zippers and rather suggests that intramolecular interactions between leucine zippers take place. A database search has revealed as the most significative homology a match with the human mitochondrial transcription termination factor (mTERF), a protein that also binds DNA as a monomer and contains three leucine zippers forming intramolecular interactions. These similarities, and the observation that mtDBP-binding sites contain the 3'-ends of mtRNAs coded by opposite strands and the 3'-end of the D-loop structure, point to a dual function of the protein in modulating sea urchin mitochondrial DNA transcription and replication.

Mitochondrial gene expression has been investigated in synaptic endings from rat cerebral cortex isolated at various stages during the postnatal development and maturation of the animal. The pattern of the mitochondrial translation products labeled in vitro in rat brain synaptosomes revealed some distinctive features when compared with the pattern observed in a rat fibroblast cell line, the most remarkable being the apparent absence of labeling of the ND5 product. This absence contrasted with the presence in synaptosomes of an amount of ND5 mRNA comparable with that found in the rat fibroblast cell line. The rate of mitochondrial protein synthesis per unit amount of mtDNA in brain synaptosomes showed a characteristic reproducible burst at 10-13 days after birth, thereafter declining sharply in the 3rd week to reach a level that remained constant over a 2-year period. The postnatal burst of mitochondrial protein synthesis coincided with a sharp increase in cytochrome c oxidase activity, pointing to a phase of rapid assembly of respiratory complexes. A comparison of the levels of mitochondrial mRNAs with the corresponding rates of protein synthesis during the animal development and maturation showed a lack of correlation. These observations, together with the apparent lack of translation of the ND5 mRNA, indicate that translational control plays a major role in the regulation of gene expression in rat brain synaptic mitochondria.

The unique compactness and economy of gene organization o f the mammalian mitochondrial genome and its very high CCJPY number demand that the regulation of expression of this genome follow unusual rules. Work over the past 10 years in our and other laboratories has identified some of the mechanisms operating in the control of mitochondrial gene expression in mammalian cells in culture and in differentiated mammalian cell systems. As a consequence, a general picture has begun t o emerge concerning the mechanisms by which mammalian cells adapt t o the changing energetic demands in response to functional, developmental and pathological factors.

A new human zinc finger DNA-binding protein was identified by using a yeast one-hybrid selection system. Two versions of the cDNA, encoding the same protein, were detected that differ for a 584 bp extension at the 5′ region. Sequence analysis showed that the longer clone is a full length version containing part of the 5′ untranslated region. The smaller version was fused in frame with the yeast GAL4 activation domain whereas the 5′ region of the longer clone displayed a stop codon interrupting the fusion with the GAL4 domain. Nevertheless, this clone activated the yeast HIS3 reporter gene with the same efficiency as the smaller version. Sequence comparison of the derived protein with the database showed that it belongs to a family of zinc finger DNA-binding proteins which regulate the expression of genes involved in cell proliferation. Expression of the protein in an in vitro system, DNA-binding studies and genetic experiments identify this factor as a new zinc finger DNA-binding protein which binds GC-rich sequences and contains a domain probably functioning as a transcriptional activator. The new human protein identified in this study was therefore named GC-box-binding zinc finger protein).

A quantitative study on the concentration of mitochondrial DNA and two species of mtRNA, the ribosomal (16S rRNA) and messenger (CoI mRNA) has been carried out in rat liver between -3 and 14 days of age. The cellular content of mitochondrial DNA begins to increase at one day of life and goes up linearly until 14 days of age. The cellular level of 16S rRNA and CoI mRNA changes during development: the 16S rRNA increases linearly after birth, whereas CoI mRNA shows a peak at birth and thereafter remains more or less constant. The concentration of 16S rRNA per mitochondrial DNA molecule remains substantially unchanged during development, whereas that of CoI mRNA increases before birth and, at birth, reaches values higher than in adults. These results support an independent regulation of mitochondrial rRNA and mRNA level in rat liver mitochondria during development.