The Experts below are selected from a list of 4278 Experts worldwide ranked by ideXlab platform
Nilsgoran Larsson - One of the best experts on this subject based on the ideXlab platform.
-
the amino terminal extension of mammalian mitochondrial rna polymerase ensures promoter specific transcription initiation
Nucleic Acids Research, 2014Co-Authors: Viktor Posse, Nilsgoran Larsson, Anke Dierckx, Emily Hoberg, Saba Shahzad, Camilla Koolmeister, Marcus L Wilhelmsson, Martin B Hallberg, Claes M GustafssonAbstract:Mammalian mitochondrial transcription is executed by a single subunit mitochondrial RNA polymerase (Polrmt) and its two accessory factors, mitochondrial transcription factors A and B2 (TFAM and Tfb2m). Polrmt is structurally related to single-subunit phage RNA polymerases, but it also contains a unique N-terminal extension (NTE) of unknown function. We here demonstrate that the NTE functions together with TFAM to ensure promoter-specific transcription. When the NTE is deleted, Polrmt can initiate transcription in the absence of TFAM, both from promoters and non-specific DNA sequences. Additionally, when in presence of TFAM and a mitochondrial promoter, the NTE-deleted mutant has an even higher transcription activity than wild-type polymerase, indicating that the NTE functions as an inhibitory domain. Our studies lead to a model according to which TFAM specifically recruits wild-type Polrmt to promoter sequences, relieving the inhibitory effect of the NTE, as a first step in transcription initiation. In the second step, Tfb2m is recruited into the complex and transcription is initiated.
-
Mammalian transcription factor A is a core component of the mitochondrial transcription machinery
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Yonghong Shi, Maria Falkenberg, Nilsgoran Larsson, Sjoerd Wanrooij, Anke Dierckx, Paulina H. Wanrooij, L. Marcus Wilhelmsson, Claes M GustafssonAbstract:Transcription factor A (TFAM) functions as a DNA packaging factor in mammalian mitochondria. TFAM also binds sequence-specifically to sites immediately upstream of mitochondrial promoters, but there are conflicting data regarding its role as a core component of the mitochondrial transcription machinery. We here demonstrate that TFAM is required for transcription in mitochondrial extracts as well as in a reconstituted in vitro transcription system. The absolute requirement of TFAM can be relaxed by conditions that allow DNA breathing, i.e., low salt concentrations or negatively supercoiled DNA templates. The situation is thus very similar to that described in nuclear RNA polymerase II-dependent transcription, in which the free energy of supercoiling can circumvent the need for a subset of basal transcription factors at specific promoters. In agreement with these observations, we demonstrate that TFAM has the capacity to induce negative supercoils in DNA, and, using the recently developed nucleobase analog FRET-pair tC(O)-tC(nitro), we find that TFAM distorts significantly the DNA structure. Our findings differ from recent observations reporting that TFAM is not a core component of the mitochondrial transcription machinery. Instead, our findings support a model in which TFAM is absolutely required to recruit the transcription machinery during initiation of transcription.
-
increased mitochondrial ca2 and decreased sarcoplasmic reticulum ca2 in mitochondrial myopathy
Human Molecular Genetics, 2009Co-Authors: Jan Aydin, Chan Bae Park, Nilsgoran Larsson, Daniel C Andersson, Sandra L Hanninen, Anna Wredenberg, Pasi Tavi, Joseph D Bruton, Hakan WesterbladAbstract:Genetic mutations that affect mitochondrial function often cause skeletal muscle dysfunction. Here, we used mice with skeletal-muscle-specific disruption of the nuclear gene for mitochondrial transcription factor A (TFAM) to study whether changes in cellular Ca 2+ handling is part of the mechanism of muscle dysfunction in mitochondrial myopathy. Force measurements were combined with measurements of cytosolic Ca 2+ , mitochondrial Ca 2+ and membrane potential and reactive oxygen species in intact, adult muscle fibres. The results show reduced sarcoplasmic reticulum (SR) Ca 2+ storage capacity in TFAM KO muscles due to a decreased expression of calsequestrin-1. This resulted in decreased SR Ca 2+ release during contraction and hence lower force production in TFAM KO than in control muscles. Additionally, there were no signs of oxidative stress in TFAM KO cells, whereas they displayed increased mitochondrial [Ca 2+ ] during repeated contractions. Mitochondrial [Ca 2+ ] remained elevated long after the end of stimulation in muscle cells from terminally ill TFAM KO mice, and the increase was smaller in the presence of the cyclophilin D-binding inhibitor cyclosporin A. The mitochondrial membrane potential in TFAM KO cells did not decrease during repeated contractions. In conclusion, we suggest that the observed changes in Ca 2+ handling are adaptive responses with long-term detrimental effects. Reduced SR Ca 2+ release likely decreases ATP expenditure, but it also induces muscle weakness. Increased [Ca 2+ ] mit will stimulate mitochondrial metabolism acutely but may also trigger cell damage.
-
the mitochondrial rna polymerase contributes critically to promoter specificity in mammalian cells
The EMBO Journal, 2004Co-Authors: Martina Gaspari, Maria Falkenberg, Nilsgoran Larsson, Claes M GustafssonAbstract:Initiation of transcription in mammalian mitochondria depends on three proteins: mitochondrial RNA polymerase (POLRMT), mitochondrial transcription factor A (TFAM) and mitochondrial transcription factor B2 (TFB2M). We show here that the recombinant mouse and human transcription machineries are unable to initiate transcription in vitro from the heterologous light-strand promoter (LSP) of mitochondrial DNA. This species specificity is dependent on the interaction of TFAM and POLRMT with specific distal and proximal promoter elements. A sequence element localized from position −1 to −2 relative to the transcription start site in LSP functionally interacts with POLRMT. The POLRMT/TFB2M heterodimer is unable to interact with promoter elements and initiate even abortive transcription in the absence of TFAM. TFAM is thus an integral part of the mammalian transcription machinery, and we propose that TFAM induces a structural change of the promoter that is required for POLRMT-dependent promoter recognition.
-
mitochondrial transcription factor a regulates mtdna copy number in mammals
Human Molecular Genetics, 2004Co-Authors: Mats I Ekstrand, Martina Gaspari, Maria Falkenberg, Anja Rantanen, Chan Bae Park, Kjell Hultenby, Pierre Rustin, Claes M Gustafsson, Nilsgoran LarssonAbstract:Mitochondrial DNA (mtDNA) copy number regulation is altered in several human mtDNA-mutation diseases and it is also important in a variety of normal physiological processes. Mitochondrial transcription factor A (TFAM) is essential for human mtDNA transcription and we demonstrate here that it is also a key regulator of mtDNA copy number. We initially performed in vitro transcription studies and determined that the human TFAM protein is a poor activator of mouse mtDNA transcription, despite its high capacity for unspecific DNA binding. Next, we generated P1 artificial chromosome (PAC) transgenic mice ubiquitously expressing human TFAM. The introduced human TFAM gene was regulated in a similar fashion as the endogenous mouse TFAM gene and expression of the human TFAM protein in the mouse did not result in down-regulation of the endogenous expression. The PAC-TFAM mice thus had a net overexpression of TFAM protein and this resulted in a general increase of mtDNA copy number. We used a combination of mice with TFAM overexpression and TFAM knockout and demonstrated that mtDNA copy number is directly proportional to the total TFAM protein levels also in mouse embryos. Interestingly, the expression of human TFAM in the mouse results in up-regulation of mtDNA copy number without increasing respiratory chain capacity or mitochondrial mass. It is thus possible to experimentally dissociate mtDNA copy number regulation from mtDNA expression and mitochondrial biogenesis in mammals in vivo. In conclusion, our results provide genetic evidence for a novel role for TFAM in direct regulation of mtDNA copy number in mammals.
Claes M Gustafsson - One of the best experts on this subject based on the ideXlab platform.
-
in vitro reconstituted nucleoids can block mitochondrial dna replication and transcription
Cell Reports, 2014Co-Authors: Geraldine Farge, Majda Mehmedovic, Sie M J L Van Den Wildenberg, Gijs J L Wuite, Claes M Gustafsson, Marian Baclayon, Wouter H Roos, Maria FalkenbergAbstract:The mechanisms regulating the number of active copies of mtDNA are still unclear. A mammalian cell typically contains 1,000-10,000 copies of mtDNA, which are packaged into nucleoprotein complexes termed nucleoids. The main protein component of these structures is mitochondrial transcription factor A (TFAM). Here, we reconstitute nucleoid-like particles in vitro and demonstrate that small changes in TFAM levels dramatically impact the fraction of DNA molecules available for transcription and DNA replication. Compaction by TFAM is highly cooperative, and at physiological ratios of TFAM to DNA, there are large variations in compaction, from fully compacted nucleoids to naked DNA. In compacted nucleoids, TFAM forms stable protein filaments on DNA that block melting and prevent progression of the replication and transcription machineries. Based on our observations, we suggest that small variations in the TFAM-to-mtDNA ratio may be used to regulate mitochondrial gene transcription and DNA replication.
-
the amino terminal extension of mammalian mitochondrial rna polymerase ensures promoter specific transcription initiation
Nucleic Acids Research, 2014Co-Authors: Viktor Posse, Nilsgoran Larsson, Anke Dierckx, Emily Hoberg, Saba Shahzad, Camilla Koolmeister, Marcus L Wilhelmsson, Martin B Hallberg, Claes M GustafssonAbstract:Mammalian mitochondrial transcription is executed by a single subunit mitochondrial RNA polymerase (Polrmt) and its two accessory factors, mitochondrial transcription factors A and B2 (TFAM and Tfb2m). Polrmt is structurally related to single-subunit phage RNA polymerases, but it also contains a unique N-terminal extension (NTE) of unknown function. We here demonstrate that the NTE functions together with TFAM to ensure promoter-specific transcription. When the NTE is deleted, Polrmt can initiate transcription in the absence of TFAM, both from promoters and non-specific DNA sequences. Additionally, when in presence of TFAM and a mitochondrial promoter, the NTE-deleted mutant has an even higher transcription activity than wild-type polymerase, indicating that the NTE functions as an inhibitory domain. Our studies lead to a model according to which TFAM specifically recruits wild-type Polrmt to promoter sequences, relieving the inhibitory effect of the NTE, as a first step in transcription initiation. In the second step, Tfb2m is recruited into the complex and transcription is initiated.
-
Mammalian transcription factor A is a core component of the mitochondrial transcription machinery
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Yonghong Shi, Maria Falkenberg, Nilsgoran Larsson, Sjoerd Wanrooij, Anke Dierckx, Paulina H. Wanrooij, L. Marcus Wilhelmsson, Claes M GustafssonAbstract:Transcription factor A (TFAM) functions as a DNA packaging factor in mammalian mitochondria. TFAM also binds sequence-specifically to sites immediately upstream of mitochondrial promoters, but there are conflicting data regarding its role as a core component of the mitochondrial transcription machinery. We here demonstrate that TFAM is required for transcription in mitochondrial extracts as well as in a reconstituted in vitro transcription system. The absolute requirement of TFAM can be relaxed by conditions that allow DNA breathing, i.e., low salt concentrations or negatively supercoiled DNA templates. The situation is thus very similar to that described in nuclear RNA polymerase II-dependent transcription, in which the free energy of supercoiling can circumvent the need for a subset of basal transcription factors at specific promoters. In agreement with these observations, we demonstrate that TFAM has the capacity to induce negative supercoils in DNA, and, using the recently developed nucleobase analog FRET-pair tC(O)-tC(nitro), we find that TFAM distorts significantly the DNA structure. Our findings differ from recent observations reporting that TFAM is not a core component of the mitochondrial transcription machinery. Instead, our findings support a model in which TFAM is absolutely required to recruit the transcription machinery during initiation of transcription.
-
the mitochondrial rna polymerase contributes critically to promoter specificity in mammalian cells
The EMBO Journal, 2004Co-Authors: Martina Gaspari, Maria Falkenberg, Nilsgoran Larsson, Claes M GustafssonAbstract:Initiation of transcription in mammalian mitochondria depends on three proteins: mitochondrial RNA polymerase (POLRMT), mitochondrial transcription factor A (TFAM) and mitochondrial transcription factor B2 (TFB2M). We show here that the recombinant mouse and human transcription machineries are unable to initiate transcription in vitro from the heterologous light-strand promoter (LSP) of mitochondrial DNA. This species specificity is dependent on the interaction of TFAM and POLRMT with specific distal and proximal promoter elements. A sequence element localized from position −1 to −2 relative to the transcription start site in LSP functionally interacts with POLRMT. The POLRMT/TFB2M heterodimer is unable to interact with promoter elements and initiate even abortive transcription in the absence of TFAM. TFAM is thus an integral part of the mammalian transcription machinery, and we propose that TFAM induces a structural change of the promoter that is required for POLRMT-dependent promoter recognition.
-
mitochondrial transcription factor a regulates mtdna copy number in mammals
Human Molecular Genetics, 2004Co-Authors: Mats I Ekstrand, Martina Gaspari, Maria Falkenberg, Anja Rantanen, Chan Bae Park, Kjell Hultenby, Pierre Rustin, Claes M Gustafsson, Nilsgoran LarssonAbstract:Mitochondrial DNA (mtDNA) copy number regulation is altered in several human mtDNA-mutation diseases and it is also important in a variety of normal physiological processes. Mitochondrial transcription factor A (TFAM) is essential for human mtDNA transcription and we demonstrate here that it is also a key regulator of mtDNA copy number. We initially performed in vitro transcription studies and determined that the human TFAM protein is a poor activator of mouse mtDNA transcription, despite its high capacity for unspecific DNA binding. Next, we generated P1 artificial chromosome (PAC) transgenic mice ubiquitously expressing human TFAM. The introduced human TFAM gene was regulated in a similar fashion as the endogenous mouse TFAM gene and expression of the human TFAM protein in the mouse did not result in down-regulation of the endogenous expression. The PAC-TFAM mice thus had a net overexpression of TFAM protein and this resulted in a general increase of mtDNA copy number. We used a combination of mice with TFAM overexpression and TFAM knockout and demonstrated that mtDNA copy number is directly proportional to the total TFAM protein levels also in mouse embryos. Interestingly, the expression of human TFAM in the mouse results in up-regulation of mtDNA copy number without increasing respiratory chain capacity or mitochondrial mass. It is thus possible to experimentally dissociate mtDNA copy number regulation from mtDNA expression and mitochondrial biogenesis in mammals in vivo. In conclusion, our results provide genetic evidence for a novel role for TFAM in direct regulation of mtDNA copy number in mammals.
Maria Falkenberg - One of the best experts on this subject based on the ideXlab platform.
-
in vitro reconstituted nucleoids can block mitochondrial dna replication and transcription
Cell Reports, 2014Co-Authors: Geraldine Farge, Majda Mehmedovic, Sie M J L Van Den Wildenberg, Gijs J L Wuite, Claes M Gustafsson, Marian Baclayon, Wouter H Roos, Maria FalkenbergAbstract:The mechanisms regulating the number of active copies of mtDNA are still unclear. A mammalian cell typically contains 1,000-10,000 copies of mtDNA, which are packaged into nucleoprotein complexes termed nucleoids. The main protein component of these structures is mitochondrial transcription factor A (TFAM). Here, we reconstitute nucleoid-like particles in vitro and demonstrate that small changes in TFAM levels dramatically impact the fraction of DNA molecules available for transcription and DNA replication. Compaction by TFAM is highly cooperative, and at physiological ratios of TFAM to DNA, there are large variations in compaction, from fully compacted nucleoids to naked DNA. In compacted nucleoids, TFAM forms stable protein filaments on DNA that block melting and prevent progression of the replication and transcription machineries. Based on our observations, we suggest that small variations in the TFAM-to-mtDNA ratio may be used to regulate mitochondrial gene transcription and DNA replication.
-
Mammalian transcription factor A is a core component of the mitochondrial transcription machinery
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Yonghong Shi, Maria Falkenberg, Nilsgoran Larsson, Sjoerd Wanrooij, Anke Dierckx, Paulina H. Wanrooij, L. Marcus Wilhelmsson, Claes M GustafssonAbstract:Transcription factor A (TFAM) functions as a DNA packaging factor in mammalian mitochondria. TFAM also binds sequence-specifically to sites immediately upstream of mitochondrial promoters, but there are conflicting data regarding its role as a core component of the mitochondrial transcription machinery. We here demonstrate that TFAM is required for transcription in mitochondrial extracts as well as in a reconstituted in vitro transcription system. The absolute requirement of TFAM can be relaxed by conditions that allow DNA breathing, i.e., low salt concentrations or negatively supercoiled DNA templates. The situation is thus very similar to that described in nuclear RNA polymerase II-dependent transcription, in which the free energy of supercoiling can circumvent the need for a subset of basal transcription factors at specific promoters. In agreement with these observations, we demonstrate that TFAM has the capacity to induce negative supercoils in DNA, and, using the recently developed nucleobase analog FRET-pair tC(O)-tC(nitro), we find that TFAM distorts significantly the DNA structure. Our findings differ from recent observations reporting that TFAM is not a core component of the mitochondrial transcription machinery. Instead, our findings support a model in which TFAM is absolutely required to recruit the transcription machinery during initiation of transcription.
-
protein sliding and dna denaturation are essential for dna organization by human mitochondrial transcription factor a
Nature Communications, 2012Co-Authors: Geraldine Farge, Niels Laurens, Onno D Oekmans, Sie M J L Van Den Wildenberg, Linda Dekke, Martina Gaspari, Claes M Gustafsso, Erwi J G Peterma, Maria Falkenberg, Gijs J L WuiteAbstract:The mitochondrial transcription factor A (TFAM) mediates both mitochondrial transcription and DNA compaction, but how it achieves these two functions is unknown. In this study, TFAM is shown to slide along DNA and cause local melting, suggesting a mechanism for how TFAM modulates both transcription and compaction.
-
the mitochondrial rna polymerase contributes critically to promoter specificity in mammalian cells
The EMBO Journal, 2004Co-Authors: Martina Gaspari, Maria Falkenberg, Nilsgoran Larsson, Claes M GustafssonAbstract:Initiation of transcription in mammalian mitochondria depends on three proteins: mitochondrial RNA polymerase (POLRMT), mitochondrial transcription factor A (TFAM) and mitochondrial transcription factor B2 (TFB2M). We show here that the recombinant mouse and human transcription machineries are unable to initiate transcription in vitro from the heterologous light-strand promoter (LSP) of mitochondrial DNA. This species specificity is dependent on the interaction of TFAM and POLRMT with specific distal and proximal promoter elements. A sequence element localized from position −1 to −2 relative to the transcription start site in LSP functionally interacts with POLRMT. The POLRMT/TFB2M heterodimer is unable to interact with promoter elements and initiate even abortive transcription in the absence of TFAM. TFAM is thus an integral part of the mammalian transcription machinery, and we propose that TFAM induces a structural change of the promoter that is required for POLRMT-dependent promoter recognition.
-
mitochondrial transcription factor a regulates mtdna copy number in mammals
Human Molecular Genetics, 2004Co-Authors: Mats I Ekstrand, Martina Gaspari, Maria Falkenberg, Anja Rantanen, Chan Bae Park, Kjell Hultenby, Pierre Rustin, Claes M Gustafsson, Nilsgoran LarssonAbstract:Mitochondrial DNA (mtDNA) copy number regulation is altered in several human mtDNA-mutation diseases and it is also important in a variety of normal physiological processes. Mitochondrial transcription factor A (TFAM) is essential for human mtDNA transcription and we demonstrate here that it is also a key regulator of mtDNA copy number. We initially performed in vitro transcription studies and determined that the human TFAM protein is a poor activator of mouse mtDNA transcription, despite its high capacity for unspecific DNA binding. Next, we generated P1 artificial chromosome (PAC) transgenic mice ubiquitously expressing human TFAM. The introduced human TFAM gene was regulated in a similar fashion as the endogenous mouse TFAM gene and expression of the human TFAM protein in the mouse did not result in down-regulation of the endogenous expression. The PAC-TFAM mice thus had a net overexpression of TFAM protein and this resulted in a general increase of mtDNA copy number. We used a combination of mice with TFAM overexpression and TFAM knockout and demonstrated that mtDNA copy number is directly proportional to the total TFAM protein levels also in mouse embryos. Interestingly, the expression of human TFAM in the mouse results in up-regulation of mtDNA copy number without increasing respiratory chain capacity or mitochondrial mass. It is thus possible to experimentally dissociate mtDNA copy number regulation from mtDNA expression and mitochondrial biogenesis in mammals in vivo. In conclusion, our results provide genetic evidence for a novel role for TFAM in direct regulation of mtDNA copy number in mammals.
Hakan Westerblad - One of the best experts on this subject based on the ideXlab platform.
-
impaired aerobic capacity and premature fatigue preceding muscle weakness in the skeletal muscle TFAM knockout mouse model
Disease Models & Mechanisms, 2021Co-Authors: Benjamin Chatel, Sylvie Ducreux, Zeina Harhous, Nadia Bendridi, Isabelle Varlet, Augustin C Ogier, Monique Bernard, Julien Gondin, Jennifer Rieusset, Hakan WesterbladAbstract:Mitochondrial diseases are genetic disorders leading to an impaired mitochondrial function and resulting in exercise intolerance and muscle weakness. In patients, muscle fatigue due to defects in mitochondrial oxidative capacities commonly precedes muscle weakness. In mice, the fast-twitch skeletal muscle-specific TFAM deletion (TFAM KO) leads to deficit in the respiratory chain activity, severe muscle weakness and early death. Here, we performed a time-course study of mitochondrial and muscular dysfunctions in 11 and 14 weeks TFAM KO mice, i.e., before and when mice are about to enter the terminal stage, respectively. While force in the unfatigued state was reduced in TFAM KO mice as compared to control littermates (WT) only at 14 weeks, during repeated submaximal contractions fatigue was faster at both ages. During fatiguing stimulation, total phosphocreatine breakdown was larger in TFAM KO muscle than in WT muscle at both ages whereas phosphocreatine consumption was faster only at 14 weeks. In conclusion, the TFAM KO mouse model represents a reliable model of lethal mitochondrial myopathy where impaired mitochondrial energy production and premature fatigue occur before muscle weakness and early death.
-
increased mitochondrial ca2 and decreased sarcoplasmic reticulum ca2 in mitochondrial myopathy
Human Molecular Genetics, 2009Co-Authors: Jan Aydin, Chan Bae Park, Nilsgoran Larsson, Daniel C Andersson, Sandra L Hanninen, Anna Wredenberg, Pasi Tavi, Joseph D Bruton, Hakan WesterbladAbstract:Genetic mutations that affect mitochondrial function often cause skeletal muscle dysfunction. Here, we used mice with skeletal-muscle-specific disruption of the nuclear gene for mitochondrial transcription factor A (TFAM) to study whether changes in cellular Ca 2+ handling is part of the mechanism of muscle dysfunction in mitochondrial myopathy. Force measurements were combined with measurements of cytosolic Ca 2+ , mitochondrial Ca 2+ and membrane potential and reactive oxygen species in intact, adult muscle fibres. The results show reduced sarcoplasmic reticulum (SR) Ca 2+ storage capacity in TFAM KO muscles due to a decreased expression of calsequestrin-1. This resulted in decreased SR Ca 2+ release during contraction and hence lower force production in TFAM KO than in control muscles. Additionally, there were no signs of oxidative stress in TFAM KO cells, whereas they displayed increased mitochondrial [Ca 2+ ] during repeated contractions. Mitochondrial [Ca 2+ ] remained elevated long after the end of stimulation in muscle cells from terminally ill TFAM KO mice, and the increase was smaller in the presence of the cyclophilin D-binding inhibitor cyclosporin A. The mitochondrial membrane potential in TFAM KO cells did not decrease during repeated contractions. In conclusion, we suggest that the observed changes in Ca 2+ handling are adaptive responses with long-term detrimental effects. Reduced SR Ca 2+ release likely decreases ATP expenditure, but it also induces muscle weakness. Increased [Ca 2+ ] mit will stimulate mitochondrial metabolism acutely but may also trigger cell damage.
Mark C Williams - One of the best experts on this subject based on the ideXlab platform.
-
TFAM regulates mitochondrial transcription through sequence specific dna looping
Biophysical Journal, 2016Co-Authors: Divakaran Murugesapillai, Maria F Lodeiro, Louis J Maher, Craig E Cameron, Mark C WilliamsAbstract:Mitochondrial transcription factor A (TFAM) is an abundant human mitochondrial High Mobility Group Box (HMGB) protein. TFAM is an architectural protein that shapes mitochondrial DNA (mtDNA) and regulates mitochondrial transcription. Sequence-specific binding of TFAM to DNA upstream of the light-strand promoter (LSP) leads to bending that correlates with transactivation of this promoter. Here we use a dual promoter construct containing both LSP, HSP1 and the natural inter-promoter region (IPR) to understand how TFAM binding governs transcription activation. We show that the IPR contributes to TFAM transactivation of HSP1, while removal of the carboxyl-terminal tail of TFAM (TFAM-ΔCT26) leads to a complete loss of transactivation of HSP1, with only minimal effects on LSP, suggesting a different biophysical mechanism for TFAM-mediated transcription activation from HSP1. To understand the mechanism by which TFAM activates HSP1 transcription, we used atomic force microscopy (AFM) in liquid to probe the effect of TFAM on the IPR construct. We observe that at the low concentrations of TFAM where transcription activation is high, TFAM induces the formation of DNA loops in a region defined by the IPR, with TFAM present at the strand crossing point. Under the same conditions, TFAM-ΔCT26 fails to induce DNA looping. While random DNA also forms loops in the presence of TFAM, TFAM is twice as likely to mediate the loops when the IPR sequence is present. Optical tweezers experiments also demonstrate that TFAM stabilizes DNA loops, which require significant force to break. Taken together, our results are consistent with sequence-specific DNA looping contributing to TFAM transactivation of HSP1, suggesting that unique mechanisms are employed for TFAM-dependent transcription at LSP and HSP1.
-
26 sequence specific dna looping by mitochondrial transcription factor a TFAM
Journal of Biomolecular Structure & Dynamics, 2015Co-Authors: Divakaran Murugesapillai, Maria F Lodeiro, Craig E Cameron, James L Maher, Mark C WilliamsAbstract:Mitochondrial transcription factor A (TFAM) is an abundant human mitochondrial High Mobility Group Box (HMGB) protein. Similar to several human nuclear HMGB proteins, TFAM has two HMGB domains that...
