Sea urchin mtDBP is a two-faced transcription termination factor with a biased polarity depending on the RNA polymerase.

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.

Iron(II) induces changes in the conformation of mammalian mitochondrial DNA resulting in a reduction of its transcriptional rate.

Living isolated mitochondria incubated with iron(II) show a major alteration in mitochondrial DNA (mtDNA) conformational forms as assessed by Southern blot analysis of undigested mtDNA. In the presence of iron(II), form I is transformed into form III in a dose-dependent manner. This alteration in mtDNA conformation shows a strong correlation with a decrease in the mtDNA transcription rate (r=0.965, P < 0.002), suggesting that iron(II) load results in double-strand breaks and unwinding of mtDNA, which, in turn, is unable to maintain its normal transcriptional rate.

The mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episode syndrome-associated human mitochondrial tRNALeu(UUR) mutation causes aminoacylation deficiency and concomitant reduced association of mRNA with ribosomes.

The pathogenetic mechanism of the mitochondrial tRNA(Leu(UUR)) A3243G transition associated with the mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome has been investigated in transmitochondrial cell lines constructed by transfer of mutant mitochondrial DNA (mtDNA)-carrying mitochondria from three genetically unrelated MELAS patients or of isogenic wild-type mtDNA-carrying organelles into human mtDNA-less cells. An in vivo footprinting analysis of the mtDNA segment within the tRNA(Leu(UUR)) gene that binds the transcription termination factor failed to reveal any difference in occupancy of sites or qualitative interaction with the protein between mutant and wild-type mtDNAs. Cell lines nearly homoplasmic for the mutation exhibited a strong (70-75%) reduction in the level of aminoacylated tRNA(Leu(UUR)) and a decrease in mitochondrial protein synthesis rate. The latter, however, did not show any significant correlation between synthesis defect of the individual polypeptides and number or proportion of UUR codons in their mRNAs, suggesting that another step, other than elongation, may be affected. Sedimentation analysis in sucrose gradient showed a reduction in size of the mitochondrial polysomes, while the distribution of the two rRNA components and of the mRNAs revealed decreased association of mRNA with ribosomes and, in the most affected cell line, pronounced degradation of the mRNA associated with slowly sedimenting structures. Therefore, several lines of evidence indicate that the protein synthesis defect in A3243G MELAS mutation-carrying cells is mainly due to a reduced association of mRNA with ribosomes, possibly as a consequence of the tRNA(Leu(UUR)) aminoacylation defect.

[Recent advances in genetics of epilepsy. Genetic of mitochondrial epilepsy]. / Avances recientes en genética de las epilepsias. Genética de las epilepsias mitocondriales.

INTRODUCTION: Recently the molecular basis of a series of clinical disorders associated with defects in the oxidative phosphorylation system (OXPHOS system) leading to ATP synthesis, the final pathway of mitochondrial energy metabolism, has been established. The polypeptide components of the OXPHOS system are codified in both nuclear and mitochondrial DNA. Therefore these mitochondrial diseases may be originated by mutations of genes found in both genetic systems. DEVELOPMENT: In recent years, several such neuromuscular diseases have been defined and associated with mitochondrial DNA mutations. One of the most striking of these is the syndrome of myoclonic epilepsy with ragged red fibres (MERRF), characterized by myoclonic epilepsy of maternal inheritance. This disorder is caused by a specific mutation on the mitochondrial tRNA(Lys) (position 8344), which gives rise to a reduction in the level of lysil-tRNA(Lys) and thus to premature termination of the translation of proteins codified in the mitochondrial DNA.

Genética de las epilepsias mitocondriales / The genetics of mitochondrial epilepsy

Introducción. Recientemente se ha establecido la base molecular de una serie de trastornos clínicos asociados a defectos en el sistema de fosforilación oxidativa (sistema OXPHOS) que conduce a la síntesis de ATP, la ruta final del metabolismo energético mitocondrial. Los polipéptidos componentes del sistema OXPHOS están codificados tanto en el ADN nuclear como en el mitocondrial, por lo que estas enfermedades mitocondriales pueden estar originadas por mutaciones en genes localizados en ambos sistemas genéticos. Desarrollo. En los últimos años se han definido y asociado varias de estas enfermedades neuromusculares con mutaciones en el ADN mitocondrial. Entre ellas, una de las que más ha llamado la atención es el síndrome de epilepsia mioclónica con fibras rojo-rasgadas (MERRF), caracterizado por epilepsias mioclónicas y por presentar un tipo de herencia materna. Esta enfermedad está causada por una mutación puntual en el ARNtLys mitocondrial (posición 8344), que origina una disminución de los niveles de lisil-ARNtLys y, por consiguiente, una terminación prematura de la traducción de las proteínas codificadas en el ADN mitocondrial (AU)

Autonomous regulation in mammalian mitochondrial DNA transcription.

The regulation of the oxidative phosphorylation system (OXPHOS) biogenesis in eukaryotic cells is unique since it involves the expression of two genomes, the mitochondrial DNA (mtDNA) and the nuclear DNA (nDNA). The considerable effort done in collecting information on the factors that influence the expression of the genes encoded in mtDNA and nDNA has revealed that a multiplicity of regulatory options are available in mammalian cells to perform this task. Thus, at least three archetypal situations can be distinguished: mitochondrial proliferation, mitochondrial differentiation, and mitochondrial local tuning (MLT). Each of them seems to be predominantly under the control of specific strategies of regulation, although the description of the detailed molecular mechanisms involved is still in its beginnings. In the present review, we focus on the evidence supporting the existence of mechanisms for autonomous regulation of mtDNA transcription and its role in the integrated regulation of the OXPHOS system biogenesis.

Direct regulation of mitochondrial RNA synthesis by thyroid hormone.

We have analyzed the influence of in vivo treatment and in vitro addition of thyroid hormone on in organello mitochondrial DNA (mtDNA) transcription and, in parallel, on the in organello footprinting patterns at the mtDNA regions involved in the regulation of transcription. We found that thyroid hormone modulates mitochondrial RNA levels and the mRNA/rRNA ratio by influencing the transcriptional rate. In addition, we found conspicuous differences between the mtDNA dimethyl sulfate footprinting patterns of mitochondria derived from euthyroid and hypothyroid rats at the transcription initiation sites but not at the mitochondrial transcription termination factor (mTERF) binding region. Furthermore, direct addition of thyroid hormone to the incubation medium of mitochondria isolated from hypothyroid rats restored the mRNA/rRNA ratio found in euthyroid rats as well as the mtDNA footprinting patterns at the transcription initiation area. Therefore, we conclude that the regulatory effect of thyroid hormone on mitochondrial transcription is partially exerted by a direct influence of the hormone on the mitochondrial transcription machinery. Particularly, the influence on the mRNA/rRNA ratio is achieved by selective modulation of the alternative H-strand transcription initiation sites and does not require the previous activation of nuclear genes. These results provide the first functional demonstration that regulatory signals, such as thyroid hormone, that modify the expression of nuclear genes can also act as primary signals for the transcriptional apparatus of mitochondria.

Functional analysis of in vivo and in organello footprinting of HeLa cell mitochondrial DNA in relationship to ATP and ethidium bromide effects on transcription.

In vivo and in organello footprinting techniques based on methylation interference have been utilized to investigate protein-DNA interactions in the transcription initiation and rDNA transcription termination regions of human mitochondrial DNA (mtDNA) in functionally active mitochondria. In particular, the changes in methylation reactivity of these regions in response to treatment of the organelles with ATP or ethidium bromide, which affects differentially the rates of mitochondrial rRNA and mRNA synthesis, have been analyzed. Two major sites of protein-DNA interactions have been identified in the main control region of mtDNA, both in vivo and in organello, which correspond to the regions of the light-strand promoter and heavy-strand rRNA-specific promoter. The in organello footprinting of the latter showed ATP- and ethidium bromide-dependent modifications that could be correlated with changes in the rate of rRNA but not of mRNA synthesis. By contrast, no ATP effects were observed on the in organello footprinting pattern of the termination region and on in vitro transcription termination, strongly suggesting that ATP control of rRNA synthesis occurs at the initiation level. Several methylation interference sites were found upstream of the whole H-strand transcription unit, pointing to possible protein-DNA interactions related to the activity of this unit. In vivo footprinting of the rDNA transcription termination region of human mtDNA has revealed a very strong protection pattern, indicating a high degree of occupancy of the termination site by mitochondrial transcription termination factor (approximately 80%).

Disorders of mitochondria and related metabolism.

Mitochondrial disorders are caused by mutations in either nuclear or mitochondrial genes involved in the synthesis of respiratory chain subunits or in their post-translational control. Molecular lesions of mitochondrial DNA are a frequent cause of defective oxidative phosphorylation. Although only one mutation of nuclear-encoded oxidative phosphorylation subunits has so far been reported in humans, numerous biochemically defined disorders are attributed to nuclear gene defects. The pathogenesis of these disorders has been investigated through a combination of different expertises, including keen clinical observation, classical biochemistry and muscle morphology, molecular and cellular biology, linkage analysis and population genetic studies.

The human mitochondrial transcription termination factor (mTERF) is a multizipper protein but binds to DNA as a monomer, with evidence pointing to intramolecular leucine zipper interactions.

The human mitochondrial transcription termination factor (mTERF) cDNA has been cloned and expressed in vitro, and two alternative precursors of the protein have been imported into isolated mitochondria and processed to the mature protein. The precursors contain a mitochondrial targeting sequence, and the mature mTERF (342 residues) exhibits three leucine zippers, of which one is bipartite, and two widely spaced basic domains. The in vitro synthesized mature protein has the expected specific binding capacity for a double-stranded oligonucleotide containing the tridecamer sequence required for directing termination, and produces a DNase I footprint very similar to that produced by the natural protein. However, in contrast to the latter, it lacks transcription termination-promoting activity in an in vitro system, pointing to another component(s) being required for making mTERF termination-competent. A detailed structure-function analysis of the recombinant protein and mutagenized versions of it by band shift assays has demonstrated that both basic domains and the three leucine zipper motifs are necessary for DNA binding. Furthermore, a variety of tests have shown that both the recombinant and the natural mTERF bind to DNA as a monomer, arguing against a dimerization role for the leucine zippers, and rather pointing, together with the results of mutagenesis experiments, to intramolecular leucine zipper interactions being required to bring the two basic domains in close register with the mTERF target DNA sequence.

The synthesis of mRNA in isolated mitochondria can be maintained for several hours and is inhibited by high levels of ATP.

The dependence for the maintenance of the synthesis and maturation of mitochondrial RNA on the supply of nucleo-cytoplasmic factors has been investigated by a novel in organello RNA synthesis system. We found that mitochondrial DNA transcription can be maintained for several hours in isolated mitochondria. Analysis of the individual mitochondrial RNA species revealed that: the processing of the rRNA precursors and the stability of the mature rRNAs, but not the transcription itself, is severely impaired after short periods of incubation, indicating that these processes are strongly dependent on the mitochondrial interaction with the nucleo-cytoplasmic compartment; the events that lead to the synthesis, processing and turnover of the mitochondrial mRNAs do not require the continuous supply of nucleo-cytoplasmic factors, that are accumulated in excess by mitochondria. Furthermore, we present evidence indicating an inhibition of high ATP levels on the mitochondrial RNA polymerase activity, both in organello and in vitro. Consequently, it is proposed that mitochondrial mRNA synthesis can be regulated in response to changes in intramitochondrial ATP levels. This regulation of mitochondrial mRNA synthesis together with their very rapid turnover described here and elsewhere [Gelfand, R. & Attardi, G. (1981) Mol. Cell Biol. 1, 497-511], could represent a mechanism that would allow each individual mitochondrion to adjust its optimal levels of mRNA, and hence its translation capacity, in response to local energetic demands.

RNA synthesis in isolated mitochondria from brain cortex, cerebellum and stem: evidence of different transcriptional rates.

1. A system for studying RNA synthesis in isolated sheep brain mitochondria was set up to investigate the transcriptional activity of different brain regions (cortex, cerebellum and brain stem). In this system, mitochondrial DNA is transcribed and RNA processed in a way that faithfully reproduces the in vivo process. 2. The comparison of the electrophoretic patterns of the mitochondrial DNA transcription products showed that although they were qualitatively similar, there were large differences in the rate of mitochondrial DNA transcription of the three regions studied, cerebellum and brain stem showing transcriptional rates which were 34 and 18% respectively of that of cerebral cortex.

Specific increase of a mitochondrial RNA transcript in chronic ethanol-fed rats.

An in vitro transcription system utilizing isolated mitochondria has been used to study the effect of chronic ethanol consumption on liver mitochondrial DNA transcription. The results obtained showed an overall increase of RNA synthesis and a dramatic accumulation of a discrete polyadenylated RNA species. This effect is a consequence of the chronic ethanol consumption since these changes do not occur when isolated control mitochondria are incubated in the presence of ethanol.

A simple procedure for recovering the denaturing effect of methylmercury in agarose gel electrophoresis.

A simple and rapid procedure for recovering the denaturing effect of methylmercuric hydroxide in agarose gel electrophoresis is described. The procedure consisted of the treatment of the commercial methylmercuric hydroxide solutions with Amberlite, a mixture of anion- and cation-exchange resins. This treatment greatly improved the resolution of RNA species when fractionated by electrophoresis through agarose-CH3HgOH slab gel.

Molecular characterization and cloning of sheep mitochondrial DNA.

Mitochondrial DNA from the liver of a single Rasa Aragonesa sheep has been isolated and characterized. The size of the genome, determined by restriction enzyme analysis, was found to be 16.58 kbp. The cleavage sites for the restriction endonucleases BamHI, HindIII, EcoRI, BglII, PvuII, BstEII and PstI were mapped, and the gene organization deduced through heterologous hybridization using different cloned fragments of the rat mitochondrial genome. Fragments representative of the entire sheep genome were cloned in plasmid vectors pGEM3Z and pUN121.

Reduced synthesis of mtRNA in isolated mitochondria of senescent rat brain.

A system for studying RNA synthesis in isolated mitochondria from rat brain was set up to investigate the mechanisms responsible for the age-dependent reduction of mtRNA content. In the presence of an appropriate incubation buffer both synaptic and non-synaptic mitochondria from cerebral hemispheres were able to synthesize and process mtRNA in a way quantitatively and qualitatively similar to the in vivo transcription. The comparison of the electrophoretic pattern of mtRNAs synthesized by adult and senescent rat showed, in the senescent rat, a 50% reduction in the mtRNA synthesis rate relative to the adult value. This indicates that the age-dependent decrease of the mtRNA content is linked to a lower efficiency of the mt transcription.