| 1. |
- Linder, Tomas, et al.
(författare)
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A family of putative transcription termination factors shared amongst metazoans and plants.
- 2005
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Ingår i: Current genetics. - : Springer Science and Business Media LLC. - 0172-8083 .- 1432-0983. ; 48:4, s. 265-9
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Tidskriftsartikel (refereegranskat)abstract
- The human mitochondrial transcription termination factor (mTERF) is involved in the regulation of transcription of the mitochondrial genome. Similarity searches and phylogenetic analysis demonstrate that mTERF is a member of large and complex protein family (the MTERF family) shared amongst metazoans and plants. Interestingly, we identify three novel MTERF genes in vertebrates, which all encode proteins with predicted mitochondrial localization. Members of the MTERF family have so far not been detected in fungi, supporting the notion that mitochondrial transcription regulation may have evolved separately in yeast and animal cells.
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| 2. |
- Pellegrini, Mina
(författare)
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Molecular mechanisms of mammalian mitochondrial DNA replication and transcription
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mitochondria are cytoplasmic organelles in eukaryotic cells, mainly devoted to the synthesis of ATP, through the Oxydative Phospshorylation System (OXPHOS). The organelle has its own genome, the mitochondrial DNA (mtDNA) that encodes for all the RNA components of the mitochondrial translation apparatus (two rRNAs and 22 tRNAs) and for 13 subunits of the OXPHOS complexes. Many trans-acting factors involved in the replication and expression of the mtDNA have been identified, but the molecular mechanisms regulating these processes are still poorly understood. In our work, we have reconstituted the minimal mammalian mtDNA replisome in vitro. Together, the mitochondrial DNA polymerase (POLgamma) and the mitochondrial DNA helicase TWINKLE form a processive replication machinery, which can use doublestranded DNA (dsDNA) as template to synthesize single-stranded DNA (ssDNA) molecules of about 2 kb. The addition of the mitochondrial ssDNA-binding protein (mtSSB) stimulates the reaction, generating DNA products of about 16 kb, the size of the mammalian mtDNA molecule. These findings provide biochemical evidence for TWINKLE being the helicase at the mtDNA replication fork and define the minimal mitochondrial replisome. Previous studies had shown that the human mitochondrial transcription termination factor (mTERF) governs site-specific termination of the mitochondrial transcription process. We used bioinformatic analysis and could demonstrate that mTERF is a member of a protein family (the MTERF family) shared among metazoans and plants. Interestingly, we identified three novel MTERF genes in vertebrates (MTERF2 - 4), which all encode proteins with predicted mitochondrial localization. To analyze the function of one of these MTERF proteins, MTERF3, we used a combination of mouse genetics and biochemistry. We could demonstrate that MTERF3 is a negative regulator of mtDNA transcription initiation. The MTERF3 gene is essential because homozygous knockout mouse embryos die in midgestation. Heart-specific disruption of MTERF3 gene impairs mtDNA transcription and causes severe respiratory chain deficiency. Biochemical evidences show that MTERF3 binds the mtDNA promoter region and depletion of MTERF3 increases transcription initiation on both mtDNA strands. MTERF3 was the first described mitochondrial protein that specifically repressed mammalian mtDNA transcription initiation in vivo. A biochemical approach has been used to characterize another member of the MTERF family, MTERF2. We have confirmed the mitochondrial localization of the protein and the position of the cleavage site of its mitochondrial targeting peptide was identified. MTERF2 is a monomer in isolation and it binds mtDNA in a non sequence-specific manner. In vivo quantification experiments show that MTERF2 is relatively abundant, with one monomer present per ∼ 265 bp of mtDNA. Using formaldehyde cross-linking we demonstrated that MTERF2 is present in nucleoids, and therefore located in close proximity to mtDNA. The functional role of MTERF2 is still unknown.
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| 3. |
- Pellegrini, Mina, et al.
(författare)
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MTERF2 is a nucleoid component in mammalian mitochondria.
- 2009
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 0005-2728. ; 1787:5, s. 296-302
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Tidskriftsartikel (refereegranskat)abstract
- The mammalian MTERF family of proteins has four members, named MTERF1 to MTERF4, which were identified in homology searches using the mitochondrial transcription termination factor, mTERF (here denoted MTERF1) as query. MTERF1 and MTERF3 are known to participate in the control of mitochondrial DNA transcription, but the function of the other two proteins is not known. We here investigate the structure and function of MTERF2. Protein import experiments using isolated organelles confirm that MTERF2 is a mitochondrial protein. Edman degradation of MTERF2 isolated from stably transfected HeLa cells demonstrates that mature MTERF2 lacks a targeting peptide (amino acids 1-35) present in the precursor form of the protein. MTERF2 is a monomer in isolation and displays a non sequence-specific DNA-binding activity. In vivo quantification experiments demonstrate that MTERF2 is relatively abundant, with one monomer present per approximately 265 bp of mtDNA. In comparison, the mtDNA packaging factor TFAM is present at a ratio of one molecule per approximately 10-12 bp of mtDNA. Using formaldehyde cross-linking we demonstrate that MTERF2 is present in nucleoids, and therefore must be located in close proximity to mtDNA. Taken together, our work provides a basic biochemical characterization of MTERF2, paving the way for future functional studies.
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