Biomedical subjects

Géraldine Farge

Publications and source records attributed to Géraldine Farge.

4 recordsLinked to original sources

PubMed2006

Conserved sequence box II directs transcription termination and primer formation in mitochondria.

The human mitochondrial transcription machinery generates the RNA primers needed for initiation of heavy strand DNA synthesis. Most DNA replication events from the heavy strand origin are prematurely terminated, forming a persistent RNA-DNA hybrid, which remains annealed to the parental DNA strand. This triple-stranded structure is called the D-loop and encompasses the conserved sequence box II, a DNA element required for proper primer formation. We here use a purified recombinant mitochondrial transcription system and demonstrate that conserved sequence box II is a sequence-dependent transcription termination element in vitro. Transcription from the light strand promoter is prematurely terminated at positions 300-282 in the mitochondrial genome, which coincide with the major RNA-DNA transition points in the D-loop of human mitochondria. Based on our findings, we propose a model for primer formation at the origin of heavy strand DNA replication.

Base Sequence↗

PubMed2005

The human mitochondrial transcription termination factor (mTERF) is fully active in vitro in the non-phosphorylated form.

The human mitochondrial transcription termination factor (mTERF) is a 39-kDa protein that terminates transcription at the 3'-end of the 16 S rRNA gene and thereby controls expression of the ribosomal transcription unit of mitochondrial DNA. The transcription termination activity of human mTERF has been notoriously difficult to study in vitro, and it has been suggested that the activity of the protein is regulated by posttranslational modifications or by protein polymerization. We here characterize the activity of recombinant human mTERF expressed in insect cells. We observed that mTERF efficiently promotes sequence-specific termination in a completely recombinant and highly purified in vitro system for mitochondrial transcription. The termination activity has a distinct polarity, and we observed complete transcription termination when the mTERF-binding site is oriented in a forward position relative the heavy strand promoter but only partial transcription termination when the binding site is in the reverse position. We analyzed the biochemical characteristics of the active mTERF protein and found that it is a stable monomer at physiological salt concentration. Structural analysis, including phosphostaining, two-dimensional electrophoresis, and electrospray mass spectrometry, detected no evidence of phosphorylation. We conclude that the monomeric human mTERF is fully active in its non-phosphorylated form and that the protein does not require additional cellular factors to terminate mitochondrial transcription in vitro.

Animals↗

PubMed2004

Coordinated decrease of the expression of the mitochondrial and nuclear complex I genes in a mitochondrial mutant of Drosophila.

We have studied a mutant strain of Drosophila in which 80% of the mitochondrial DNA molecules have lost over 30% of their coding region through deletion. This deletion affects genes encoding five subunits of complex I of the respiratory chain (NADH:ubiquinone oxidoreductase). The enzymatic activity of complex I in the mutant strain is half that in the wild strain, but ATP synthesis is unaffected. The drop in enzymatic activity of complex I in the mutant strain is associated with a 50% decrease in the quantity of constitutive proteins of the complex. Moreover, in the mutant strain there is a 50% decrease in the steady-state concentration of the transcripts of the mitochondrial genes affected by the deletion. This decrease is also observed for the transcripts of the nuclear genes coding for the subunits of complex I. These results suggest a coordination of the expression of the mitochondrial and nuclear genes coding for mitochondrial proteins.

Animals↗

PubMed2002

The nuclear genome is involved in heteroplasmy control in a mitochondrial mutant strain of Drosophila subobscura.

Most (78%) mitochondrial genomes in the studied mutant strain of Drosophila subobscura have undergone a large-scale deletion (5 kb) in the coding region. This mutation is stable, and is transmitted intact to the offspring. This animal model of major rearrangements of mitochondrial genomes can be used to analyse the involvement of the nuclear genome in the production and maintenance of these rearrangements. Successive backcrosses between mutant strain females and wild-type males yield a biphasic change in heteroplasmy level: (a) a 5% decrease in mutated genomes per generation (from 78 to 55%), until the nuclear genome is virtually replaced by the wild-type genome (seven to eight crosses); and (b) a continuous decrease of 0.5% per generation when the nuclear context is completely wild-type. In parallel with these changes, NADH dehydrogenase activity, which is halved in the mutant strain (five subunits of this complex are affected by the mutation), gradually increases and stabilizes near the wild-type activity. A return to a nuclear context is accompanied by the opposite phenomena: progressive increase in heteroplasmy level and stabilization at the value seen in the wild-type strain and a decrease in the activity of complex I. These results indicate that the nuclear genome plays an important role in the control of heteroplasmy level and probably in the production of rearranged genomes.

Animals↗