RESUMEN
Mitochondrial transcription factor A (TFAM) stimulates transcription from mitochondrial DNA (mtDNA) promoters in vitro and in organello. To investigate whether changes of TFAM levels also modulate transcription and replication in situ, the protein was transiently overexpressed in cultured cells. Mitochondrial mRNAs were significantly elevated at early time points, when no expansion of the TFAM pool was yet observed, but were decreased when TFAM levels had doubled, resemb-ling in vitro results. HEK cells contain about 35 molecules of TFAM per mtDNA. High levels of TFAM were not associated with increases of full-length mtDNA, but nucleic acid species sensitive to RNAse H increased. Stimulation of transcription was more evident when TFAM was transiently overexpressed in cells pre-treated with ethidium bromide (EBr) having lowered mtDNA, TFAM and mitochondrial transcript levels. EBr rapidly inhibited mtDNA transcription, while decay of mtDNA was delayed and preferentially slowly migrating, relaxed mtDNA species were depleted. In conclusion, we show that transcription of mtDNA is submaximal in cultured cells and that a subtle increase of TFAM within the matrix is sufficient to stimulate mitochondrial transcription. Thus, this protein meets all criteria for being a key factor regulating mitochondrial transcription in vivo, but other factors are necessary for increasing mtDNA copy number, at least in cultured cells.
Asunto(s)
Replicación del ADN , ADN Mitocondrial/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Línea Celular , ADN Mitocondrial/análisis , ADN Mitocondrial/biosíntesis , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/genética , Etidio/farmacología , Células HeLa , Humanos , Mitocondrias/genética , Proteínas Mitocondriales/análisis , Proteínas Mitocondriales/genética , Proteínas Nucleares/análisis , Proteínas Nucleares/genética , Factores de Transcripción/análisis , Factores de Transcripción/genética , Transcripción Genética/efectos de los fármacos , TransfecciónRESUMEN
Mitochondrial transcription factor A (TFAM) has been shown to stimulate transcription from mitochondrial DNA promoters in vitro. In order to determine whether changes in TFAM levels also regulate RNA synthesis in situ, recombinant human precursor proteins were imported into the matrix of rat liver mitochondria. After uptake of wt-TFAM, incorporation of [alpha-32P]UTP into mitochondrial mRNAs as well as rRNAs was increased 2-fold (P < 0.05), whereas import of truncated TFAM lacking 25 amino acids at the C-terminus had no effect. Import of wt-TFAM into liver mitochondria from hypothyroid rats stimulated RNA synthesis up to 4-fold. We conclude that the rate of transcription is submaximal in freshly isolated rat liver mitochondria and that increasing intra-mitochondrial TFAM levels is sufficient for stimulation. The low transcription rate associated with the hypothyroid state observed in vivo as well as in organello seems to be a result of low TFAM levels, which can be recovered by treating animals with T3 in vivo or by importing TFAM in organello. Thus, this protein meets the criteria for being a key factor in regulating mitochondrial gene expression in vivo.