| 1. |
- Asin-Cayuela, Jorge, et al.
(författare)
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The human mitochondrial transcription termination factor (mTERF) is fully active in vitro in the non-phosphorylated form.
- 2005
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Ingår i: The Journal of biological chemistry. - 0021-9258. ; 280:27, s. 25499-505
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Elmlund, Hans, et al.
(författare)
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Cryo-EM Reveals Promoter DNA Binding and Conformational Flexibility of the General Transcription Factor TFIID
- 2009
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Ingår i: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 17:11, s. 1442-1452
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Tidskriftsartikel (refereegranskat)abstract
- The general transcription factor IID (TFIID) is required for initiation of RNA polymerase II-dependent transcription at many eukaryotic promoters. TFIID comprises the TATA-binding protein (TBP) and several conserved TBP-associated factors (TAFs). Recognition of the core promoter by TFIID assists assembly of the preinitiation complex. Using cryo-electron microscopy in combination with methods for ab initio single-particle reconstruction and heterogeneity analysis, we have produced density maps of two conformational states of Schizosaccharomyces pombe TFIID, containing and lacking TBP. We report that TBP-binding is coupled to a massive histone-fold domain rearrangement. Moreover, docking of the TBP-TAF1(N-terminus) atomic structure to the THID map and reconstruction of a TAF-promoter DNA complex helps to account for TAF-dependent regulation of promoter-TBP and promoter-TAF interactions.
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| 3. |
- 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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| 4. |
- 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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| 5. |
- Wanrooij, Sjoerd, et al.
(författare)
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Human mitochondrial RNA polymerase primes lagging-strand DNA synthesis in vitro
- 2008
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Ingår i: Proceedings of The National Academy of Sciences of The United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 105:32, s. 11122-11127
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Tidskriftsartikel (refereegranskat)abstract
- The mitochondrial transcription machinery synthesizes the RNA primers required for initiation of leading-strand DNA synthesis in mammalian mitochondria. RNA primers are also required for initiation of lagging-strand DNA synthesis, but the responsible enzyme has so far remained elusive. Here, we present a series of observations that suggests that mitochondrial RNA polymerase (POLRMT) can act as lagging-strand primase in mammalian cells. POLRMT is highly processive on double-stranded DNA, but synthesizes RNA primers with a length of 25 to 75 nt on a single-stranded template. The short RNA primers synthesized by POLRMT are used by the mitochondrial DNA polymerase gamma to initiate DNA synthesis in vitro. Addition of mitochondrial single-stranded DNA binding protein (mtSSB) reduces overall levels of primer synthesis, but stimulates primer-dependent DNA synthesis. Furthermore, when combined, POLRMT, DNA polymerase gamma, the DNA helicase TWINKLE, and mtSSB are capable of simultaneous leading- and lagging-strand DNA synthesis in vitro. Based on our observations, we suggest that POLRMT is the lagging-strand primase in mammalian mitochondria.
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| 6. |
- Zhu, Xuefeng, et al.
(författare)
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Distinct differences in chromatin structure at subtelomeric X and Y' elements in budding yeast.
- 2009
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Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 4:7
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Tidskriftsartikel (refereegranskat)abstract
- In Saccharomyces cerevisiae, all ends of telomeric DNA contain telomeric repeats of (TG(1-3)), but the number and position of subtelomeric X and Y' repeat elements vary. Using chromatin immunoprecipitation and genome-wide analyses, we here demonstrate that the subtelomeric X and Y' elements have distinct structural and functional properties. Y' elements are transcriptionally active and highly enriched in nucleosomes, whereas X elements are repressed and devoid of nucleosomes. In contrast to X elements, the Y' elements also lack the classical hallmarks of heterochromatin, such as high Sir3 and Rap1 occupancy as well as low levels of histone H4 lysine 16 acetylation. Our analyses suggest that the presence of X and Y' elements govern chromatin structure and transcription activity at individual chromosome ends.
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