Drosophila mitochondrial transcription factor A (d-TFAM) is dispensable for the transcription of mitochondrial DNA in Kc167 cells.
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ABSTRACT:
SUBMITTER: Goto A
PROVIDER: S-EPMC1221649 | biostudies-other | 2001 Mar
REPOSITORIES: biostudies-other
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Project description:Mitochondrial transcription factor A (TFAM) is one of the widely studied but still incompletely understood mitochondrial protein, which plays a crucial role in the maintenance and transcription of mitochondrial DNA (mtDNA). The available experimental evidence is often contradictory in assigning the same function to various TFAM domains, partly owing to the limitations of those experimental systems. Recently, we developed the GeneSwap approach, which enables in situ reverse genetic analysis of mtDNA replication and transcription and is devoid of many of the limitations of the previously used techniques. Here, we utilized this approach to analyze the contributions of the TFAM C-terminal (tail) domain to mtDNA transcription and replication. We determined, at a single amino acid (aa) resolution, the TFAM tail requirements for in situ mtDNA replication in murine cells and established that tail-less TFAM supports both mtDNA replication and transcription. Unexpectedly, in cells expressing either C-terminally truncated murine TFAM or DNA-bending human TFAM mutant L6, HSP1 transcription was impaired to a greater extent than LSP transcription. Our findings are incompatible with the prevailing model of mtDNA transcription and thus suggest the need for further refinement.
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Project description: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.
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Project description: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.
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Project description:Mammalian cells contain genetic information in two compartments, the nucleus and the mitochondria. Mitochondrial gene expression must be coordinated with nuclear gene expression to respond to cellular energetic needs. To gain insight into the coordination between the nucleus and mitochondria, there is a need to understand the regulation of transcription of mitochondrial DNA (mtDNA). Reversible protein post-translational modifications of the mtDNA transcriptional machinery may be one way to control mtDNA transcription. Here we focus on a member of the mtDNA transcription initiation complex, mitochondrial transcription factor B2 (TFB2M). TFB2M melts mtDNA at the promoter to allow the RNA polymerase (POLRMT) to access the DNA template and initiate transcription. Three phosphorylation sites have been previously identified on TFB2M by mass spectrometry: threonine 184, serine 197, and threonine 313. Phosphomimetics were established at these positions. Proteins were purified and analyzed for their ability to bind mtDNA and initiate transcription in vitro. Our results indicate phosphorylation at threonine 184 and threonine 313 impairs promoter binding and prevents transcription. These findings provide a potential regulatory mechanism of mtDNA transcription and help clarify the importance of protein post-translational modifications in mitochondrial function.
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Project description: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.
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Project description:The initiation of protein synthesis in bacteria is ruled by three canonical factors: IF1, IF2, and IF3. This system persists in human mitochondria; however, it functions in a rather different way due to specialization and adaptation to the organellar micro-environment. We focused on human mitochondrial IF3, which was earlier studied in vitro, but no knock-out cellular models have been published up to date. In this work, we generated human HeLa cell lines deficient in the MTIF3 gene and analyzed their mitochondrial function. Despite the lack of IF3mt in these cells, they preserved functional mitochondria capable of oxygen consumption and protein synthesis; however, the translation of ATP6 mRNA was selectively decreased which compromised the assembly of ATP synthase. Together with the analogous results obtained earlier for baker's yeast mitochondrial IF3, our findings point to a functional divergence of mitochondrial initiation factors from their bacterial ancestors.
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Project description: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.
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