scholarly journals Direct Regulation of Mitochondrial RNA Synthesis by Thyroid Hormone

1999 ◽  
Vol 19 (1) ◽  
pp. 657-670 ◽  
Author(s):  
José A. Enríquez ◽  
Patricio Fernández-Silva ◽  
Nuria Garrido-Pérez ◽  
Manuel J. López-Pérez ◽  
Acisclo Pérez-Martos ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Transcription Initiation ◽  
Dimethyl Sulfate ◽  
Nuclear Genes ◽  
Regulation Of Transcription ◽  
Mitochondrial Rna ◽  
Mitochondrial Transcription ◽  
In Organello

ABSTRACT We have analyzed the influence of in vivo treatment and in vitro addition of thyroid hormone on in organello mitochondrial DNA (mtDNA) transcription and, in parallel, on the in organello footprinting patterns at the mtDNA regions involved in the regulation of transcription. We found that thyroid hormone modulates mitochondrial RNA levels and the mRNA/rRNA ratio by influencing the transcriptional rate. In addition, we found conspicuous differences between the mtDNA dimethyl sulfate footprinting patterns of mitochondria derived from euthyroid and hypothyroid rats at the transcription initiation sites but not at the mitochondrial transcription termination factor (mTERF) binding region. Furthermore, direct addition of thyroid hormone to the incubation medium of mitochondria isolated from hypothyroid rats restored the mRNA/rRNA ratio found in euthyroid rats as well as the mtDNA footprinting patterns at the transcription initiation area. Therefore, we conclude that the regulatory effect of thyroid hormone on mitochondrial transcription is partially exerted by a direct influence of the hormone on the mitochondrial transcription machinery. Particularly, the influence on the mRNA/rRNA ratio is achieved by selective modulation of the alternative H-strand transcription initiation sites and does not require the previous activation of nuclear genes. These results provide the first functional demonstration that regulatory signals, such as thyroid hormone, that modify the expression of nuclear genes can also act as primary signals for the transcriptional apparatus of mitochondria.

scholarly journals Selective labelling and inactivation of creatine kinase isoenzymes by the thyroid hormone derivative N-bromoacetyl-3,3′,5-tri-iodo-l-thyronine

1993 ◽  
Vol 291 (2) ◽  
pp. 463-472 ◽  
Author(s):  
M Wyss ◽  
T Wallimann ◽  
J Köhrle
Keyword(s):  
Creatine Kinase ◽  
Thyroid Hormone ◽  
Thyroid Hormones ◽  
Rat Heart ◽  
Covalent Modification ◽  
Regulation Of Transcription ◽  
Selective Labelling ◽  
Mitochondrial Fractions

Besides their well-known regulation of transcription by binding to nuclear receptors, thyroid hormones have been suggested to have direct effects on mitochondria. In a previous study, incubation of rat heart mitochondria with 125I-labelled N-bromoacetyl-3,3′,5-tri-iodo-L-thyronine (BrAcT3), a thyroid hormone derivative with an alkylating side chain, resulted in the selective labelling of a protein doublet around M(r) 45,000 on SDS/polyacrylamide gels [Rasmussen, Köhrle, Rokos and Hesch (1989) FEBS Lett. 255, 385-390]. Now, this protein doublet has been identified as mitochondrial creatine kinase (Mi-CK). Immunoblotting experiments with the cytoplasmic and mitochondrial fractions of rat heart, brain and liver, as well as inactivation studies with the purified chicken CK isoenzymes have further demonstrated that all four CK isoenzymes (Mia-, Mib-, M- and B-CK) are indeed selectively labelled by BrAcT3. However, in contrast with their bromoalkyl derivatives, thyroid hormones themselves did not compete for CK labelling, suggesting that not the thyroid hormone moiety but rather the bromoacetyl-driven alkylation of the highly reactive ‘essential’ thiol group of CK accounts for this selective labelling. Therefore the assumption that CK isoenzymes are thyroid-hormone-binding proteins has to be dismissed. Instead, bromoacetyl-based reagents may allow a very specific covalent modification and inactivation of CK isoenzymes in vitro and in vivo.

scholarly journals Human Mitochondrial Transcription Factor B1 Interacts with the C-Terminal Activation Region of h-mtTFA and Stimulates Transcription Independently of Its RNA Methyltransferase Activity

2003 ◽  
Vol 23 (16) ◽  
pp. 5816-5824 ◽  
Author(s):  
Vicki McCulloch ◽  
Gerald S. Shadel
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Mitochondrial Gene ◽  
Factor H ◽  
Dual Function ◽  
Mitochondrial Rna ◽  
Methyltransferase Activity ◽  
Mitochondrial Transcription

ABSTRACT A significant advancement in understanding mitochondrial gene expression is the recent identification of two new human mitochondrial transcription factors, h-mtTFB1 and h-mtTFB2. Both proteins stimulate transcription in collaboration with the high-mobility group box transcription factor, h-mtTFA, and are homologous to rRNA methyltransferases. In fact, the dual-function nature of h-mtTFB1 was recently demonstrated by its ability to methylate a conserved rRNA substrate. Here, we demonstrate that h-mtTFB1 binds h-mtTFA both in HeLa cell mitochondrial extracts and in direct-binding assays via an interaction that requires the C-terminal tail of h-mtTFA, a region necessary for transcriptional activation. In addition, point mutations in conserved methyltransferase motifs of h-mtTFB1 revealed that it stimulates transcription in vitro independently of S-adenosylmethionine binding and rRNA methyltransferase activity. Furthermore, one mutation (G65A) eliminated the ability of h-mtTFB1 to bind DNA yet did not affect transcriptional activation. These results, coupled with the observation that h-mtTFB1 and human mitochondrial RNA (h-mtRNA) polymerase can also be coimmunoprecipitated, lead us to propose a model in which h-mtTFA demarcates mitochondrial promoter locations and where h-mtTFB proteins bridge an interaction between the C-terminal tail of h-mtTFA and mtRNA polymerase to facilitate specific initiation of transcription. Altogether, these data provide important new insight into the mechanism of transcription initiation in human mitochondria and indicate that the dual functions of h-mtTFB1 can be separated.

Mechanism of regulation of transcription initiation by ppGpp. I. Effects of ppGpp on transcription initiation in vivo and in vitro

2001 ◽  
Vol 305 (4) ◽  
pp. 673-688 ◽  
Author(s):  
Melanie M. Barker ◽  
Tamas Gaal ◽  
Cathleen A. Josaitis ◽  
Richard L. Gourse
Keyword(s):  
Transcription Initiation ◽  
Regulation Of Transcription

scholarly journals Stimulation of the λ p R promoter by Escherichia coli SeqA protein requires downstream GATC sequences and involves late stages of transcription initiation

Microbiology ◽  
2006 ◽  
Vol 152 (10) ◽  
pp. 2985-2992 ◽  
Author(s):  
Robert Łyżeń ◽  
Grzegorz Wȩgrzyn ◽  
Alicja Wȩgrzyn ◽  
Agnieszka Szalewska-Pałasz
Keyword(s):  
Escherichia Coli ◽  
Transcription Initiation ◽  
Negative Regulator ◽  
Regulation Of Transcription ◽  
Chromosomal Dna ◽  
Transcription Activator ◽  
Transcription Analysis ◽  
Stimulation Of

Escherichia coli SeqA protein is a major negative regulator of chromosomal DNA replication acting by sequestration, and thus inactivation, of newly formed oriC regions. However, other activities of this protein have been discovered recently, one of which is regulation of transcription. SeqA has been demonstrated to be a specific transcription factor acting at bacteriophage λ promoters p I, p aQ and p R. While SeqA-mediated stimulation of p I and p aQ occurs by facilitating functions of another transcription activator protein, cII, a mechanism for stimulation of p R remains largely unknown. Here, it has been demonstrated that two GATC sequences, located 82 and 105 bp downstream of the p R transcription start site, are necessary for this stimulation both in vivo and in vitro. SeqA-mediated activation of p R was as effective on a linear DNA template as on a supercoiled one, indicating that alterations in DNA topology are not likely to facilitate the SeqA effect. In vitro transcription analysis demonstrated that the most important regulatory effect of SeqA in p R transcription occurs after open complex formation, namely during promoter clearance. SeqA did not influence the appearance and level of abortive transcripts or the pausing during transcription elongation. Interestingly, SeqA is one of few known prokaryotic transcription factors which bind downstream of the regulated promoter and still act as transcription activators.

scholarly journals Novel regulation of transcription initiation of the peptide IX gene of adenovirus 2.

1989 ◽  
Vol 9 (10) ◽  
pp. 4265-4271 ◽  
Author(s):  
T Matsui
Keyword(s):  
Transcriptional Repression ◽  
Transcription Initiation ◽  
Nuclear Extract ◽  
Negative Control ◽  
Tata Box ◽  
Regulation Of Transcription ◽  
Direct Initiation ◽  
Upstream Element

cis-Acting elements involved in transcription of the peptide IX (pIX) gene of adenovirus 2 were identified by using in vivo transient expression assays and two in vitro transcription systems. Deletion of either the sequence between positions -45 and -70 or the TATA box abolished the initiation of pIX gene transcription in vivo and transcription with HeLa cell nuclear extracts in vitro. These results initially suggested the presence of a positive factor acting on the upstream element. However, when proteins in the nuclear extract were fractionated by column chromatography and analyzed by reconstitution of transcription in vitro, it was found that a certain fraction could direct TATA box-dependent transcription initiation even in the absence of the upstream element. Furthermore, activity inhibiting TATA box-dependent transcription was found in the nuclear extract. In contrast, inhibition of TATA box-dependent transcription was suppressed by deletion of a downstream sequence between positions +33 and +122. These results indicate that the TATA box of the pIX gene by itself has the ability to direct initiation of constitutive transcription but that the function of this element is under negative control by a repressor acting on a downstream sequence. Thus, the upstream element of the pIX gene appears to have a novel function: suppression of the transcriptional repression exerted by a downstream sequence, leading to a net transcription activation. Possible mechanisms for transcription initiation of pIX DNA are discussed.

Novel regulation of transcription initiation of the peptide IX gene of adenovirus 2

1989 ◽  
Vol 9 (10) ◽  
pp. 4265-4271
Author(s):  
T Matsui
Keyword(s):  
Transcriptional Repression ◽  
Transcription Initiation ◽  
Nuclear Extract ◽  
Negative Control ◽  
Tata Box ◽  
Regulation Of Transcription ◽  
Direct Initiation ◽  
Upstream Element

cis-Acting elements involved in transcription of the peptide IX (pIX) gene of adenovirus 2 were identified by using in vivo transient expression assays and two in vitro transcription systems. Deletion of either the sequence between positions -45 and -70 or the TATA box abolished the initiation of pIX gene transcription in vivo and transcription with HeLa cell nuclear extracts in vitro. These results initially suggested the presence of a positive factor acting on the upstream element. However, when proteins in the nuclear extract were fractionated by column chromatography and analyzed by reconstitution of transcription in vitro, it was found that a certain fraction could direct TATA box-dependent transcription initiation even in the absence of the upstream element. Furthermore, activity inhibiting TATA box-dependent transcription was found in the nuclear extract. In contrast, inhibition of TATA box-dependent transcription was suppressed by deletion of a downstream sequence between positions +33 and +122. These results indicate that the TATA box of the pIX gene by itself has the ability to direct initiation of constitutive transcription but that the function of this element is under negative control by a repressor acting on a downstream sequence. Thus, the upstream element of the pIX gene appears to have a novel function: suppression of the transcriptional repression exerted by a downstream sequence, leading to a net transcription activation. Possible mechanisms for transcription initiation of pIX DNA are discussed.

scholarly journals POLRMT regulates the switch between replication primer formation and gene expression of mammalian mtDNA

2016 ◽  
Vol 2 (8) ◽  
pp. e1600963 ◽  
Author(s):  
Inge Kühl ◽  
Maria Miranda ◽  
Viktor Posse ◽  
Dusanka Milenkovic ◽  
Arnaud Mourier ◽  
...  
Keyword(s):  
Gene Expression ◽  
Knockout Mice ◽  
Transcription Initiation ◽  
Mitochondrial Gene ◽  
Conditional Knockout ◽  
Regulatory Role ◽  
Mitochondrial Rna ◽  
Mitochondrial Transcription ◽  
Mtdna Replication

Mitochondria are vital in providing cellular energy via their oxidative phosphorylation system, which requires the coordinated expression of genes encoded by both the nuclear and mitochondrial genomes (mtDNA). Transcription of the circular mammalian mtDNA depends on a single mitochondrial RNA polymerase (POLRMT). Although the transcription initiation process is well understood, it is debated whether POLRMT also serves as the primase for the initiation of mtDNA replication. In the nucleus, the RNA polymerases needed for gene expression have no such role. Conditional knockout of Polrmt in the heart results in severe mitochondrial dysfunction causing dilated cardiomyopathy in young mice. We further studied the molecular consequences of different expression levels of POLRMT and found that POLRMT is essential for primer synthesis to initiate mtDNA replication in vivo. Furthermore, transcription initiation for primer formation has priority over gene expression. Surprisingly, mitochondrial transcription factor A (TFAM) exists in an mtDNA-free pool in the Polrmt knockout mice. TFAM levels remain unchanged despite strong mtDNA depletion, and TFAM is thus protected from degradation of the AAA+ Lon protease in the absence of POLRMT. Last, we report that mitochondrial transcription elongation factor may compensate for a partial depletion of POLRMT in heterozygous Polrmt knockout mice, indicating a direct regulatory role of this factor in transcription. In conclusion, we present in vivo evidence that POLRMT has a key regulatory role in the replication of mammalian mtDNA and is part of a transcriptional mechanism that provides a switch between primer formation for mtDNA replication and mitochondrial gene expression.

High resolution mapping of nucleic acid containing complexes in vitro and in situ

1996 ◽  
Vol 54 ◽  
pp. 48-49
Author(s):  
D. P. Bazett-Jones ◽  
M. J. Hendzel
Keyword(s):  
Nucleic Acid ◽  
High Resolution ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Heavy Atom ◽  
Regulation Of Transcription ◽  
High Exposure ◽  
Resolution Mapping ◽  
Tata Sequence

Structural analysis of combinations of nucleosomes and transcription factors on promoter and enhancer elements is necessary in order to understand the molecular mechanisms responsible for the regulation of transcription initiation. Such complexes are often not amenable to study by high resolution crystallographic techniques. We have been applying electron spectroscopic imaging (ESI) to specific problems in molecular biology related to transcription regulation. There are several advantages that this technique offers in studies of nucleoprotein complexes. First, an intermediate level of spatial resolution can be achieved because heavy atom contrast agents are not necessary. Second, mass and stoichiometric relationships of protein and nucleic acid can be estimated by phosphorus detection, an element in much higher proportions in nucleic acid than protein. Third, wrapping or bending of the DNA by the protein constituents can be observed by phosphorus mapping of the complexes. Even when ESI is used with high exposure of electrons to the specimen, important macromolecular information may be provided. For example, an image of the TATA binding protein (TBP) bound to DNA is shown in the Figure (top panel). It can be seen that the protein distorts the DNA away from itself and much of its mass sits off the DNA helix axis. Moreover, phosphorus and mass estimates demonstrate whether one or two TBP molecules interact with this particular promoter TATA sequence.

scholarly journals POLRMT mutations impair mitochondrial transcription causing neurological disease

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Monika Oláhová ◽  
Bradley Peter ◽  
Zsolt Szilagyi ◽  
Hector Diaz-Maldonado ◽  
Meenakshi Singh ◽  
...  
Keyword(s):  
Progressive External Ophthalmoplegia ◽  
Global Developmental Delay ◽  
Disease Mechanism ◽  
Mitochondrial Transcription ◽  
Functional Studies ◽  
Mtdna Deletions ◽  
Molecular Nature ◽  
Important Disease

AbstractWhile >300 disease-causing variants have been identified in the mitochondrial DNA (mtDNA) polymerase γ, no mitochondrial phenotypes have been associated with POLRMT, the RNA polymerase responsible for transcription of the mitochondrial genome. Here, we characterise the clinical and molecular nature of POLRMT variants in eight individuals from seven unrelated families. Patients present with global developmental delay, hypotonia, short stature, and speech/intellectual disability in childhood; one subject displayed an indolent progressive external ophthalmoplegia phenotype. Massive parallel sequencing of all subjects identifies recessive and dominant variants in the POLRMT gene. Patient fibroblasts have a defect in mitochondrial mRNA synthesis, but no mtDNA deletions or copy number abnormalities. The in vitro characterisation of the recombinant POLRMT mutants reveals variable, but deleterious effects on mitochondrial transcription. Together, our in vivo and in vitro functional studies of POLRMT variants establish defective mitochondrial transcription as an important disease mechanism.

Sign in / Sign up

Export Citation Format

Share Document