Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human mitochondrial transcription factor 1 (mtTF1) has been sequenced and is a nucleus-encoded DNA binding protein of 204 amino acids (24,400 daltons). Expression of human mtTF1 in bacteria yields a protein with correct physical properties and the ability to activate mitochondrial DNA promoters. Analysis of the protein's sequence reveals no similarities to any other DNA binding proteins except for the existence of two domains that are characteristic of high mobility group (HMG) proteins. Human mtTF1 is most closely related to a DNA binding HMG-box region in hUBF, a human protein known to be important for transcription by RNA polymerase I.
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PMID:Similarity of human mitochondrial transcription factor 1 to high mobility group proteins. 203 27

Transcriptional promoters of mitochondrial DNA have diverged extensively in the course of mammalian evolution. Nevertheless, the transcriptional machinery and the overall mechanisms of transcriptional control and regulation seem to be conserved. We have compared the human and murine homologs of the major DNA-binding transcriptional activator, mitochondrial transcription factor 1 (mtTF1), with unexpected results. Both proteins have similar chromatographic and transcriptional properties and are the same size. Both recognize and bind sequences between -12 and -39 within their respective homologous promoters. However, the sequences that they recognize are markedly divergent; although the base pairs they contact are situated similarly or identically with respect to the transcriptional start site, sequence identity between the two species' contact points is less than 50%. Interestingly, the two proteins are functionally interchangeable; each can bind to the heterologous light-strand promoter and can activate transcription by the heterologous mitochondrial RNA polymerase. Thus, the RNA polymerase or some as yet undetected transcription factor, rather than mTF1, may determine the strict species specificity of mitochondrial transcription. Flexible DNA sequence recognition by mtTF1, on the other hand, may be a principal facilitating mechanism for rapid control sequence evolution.
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PMID:Flexible recognition of rapidly evolving promoter sequences by mitochondrial transcription factor 1. 262 67

We purified to near homogeneity a transcription factor from human KB cell mitochondria. This factor, designated mitochondrial transcription factor 1 (mtTF1), is required for the in vitro recognition of both major promoters of human mitochondrial DNA by the homologous mitochondrial RNA polymerase. Furthermore, it has been shown to bind upstream regulatory elements of the two major promoters. After separation from RNA polymerase by phosphocellulose chromatography, mtTF1 was chromatographed on a MonoQ anion-exchange fast-performance liquid chromatography column. Analysis of mtTF1-containing fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single major polypeptide with an Mr of approximately 25,000. Centrifugation in analytical glycerol gradients indicated a sedimentation coefficient of approximately 2.5 S, consistent with a monomeric 25-kilodalton protein. Finally, when the 25-kilodalton polypeptide was excised from a stained sodium dodecyl sulfate-polyacrylamide gel and allowed to renature, it regained DNA-binding and transcriptional stimulatory activities at both promoters. Although mtTF1 is the only mitochondrial DNA-binding transcription factor to be purified and characterized, its properties, such as a high affinity for random DNA and a weak specificity for one of its target sequences, may typify this class of regulatory proteins.
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PMID:Purification and characterization of human mitochondrial transcription factor 1. 321 Nov 48

Footprinting studies with the purine-modifying reagent dimethyl sulfate and with the single-stranded DNA probing reagent potassium permanganate were carried out in isolated mitochondria from rat liver. Dimethyl sulfate footprinting allowed the detection of protein-DNA interactions within the rat analogues of the human binding sites for the transcription termination factor mTERF and for the transcription activating factor mt-TFA. Although mTERF contacts were localized only at the boundary between the 16S rRNA/tRNA(Leu)UUR genes, multiple mtTFA contacts were detected. Contact sites were located in the light and the heavy strand promoters and, in agreement with in vitro footprinting data on human mitochondria, between the conserved sequence blocks (CSB) 1 and 2 and inside CSB-1. Potassium permanganate footprinting allowed detection of a 25-base pair region entirely contained in CSB-1 in which both strands were permanganate-reactive. No permanganate reactivity was associated with the other regions of the D-loop, including CSB-2 and -3, and with the mTERF contact site. We hypothesize that the single-stranded DNA at CSB-1 may be due to a profound helix distortion induced by mtTFA binding or be associated with a RNA polymerase pause site. In any case the location in CSB-1 of the 3' end of the most abundant replication primer and of the 5' end of the prominent D-loop DNA suggests that protein-induced DNA conformational changes play an important role in directing the transition from transcription to replication in mammalian mitochondria.
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PMID:Identification by in Organello footprinting of protein contact sites and of single-stranded DNA sequences in the regulatory region of rat mitochondrial DNA. Protein binding sites and single-stranded DNA regions in isolated rat liver mitochondria. 755 32

We have constructed a series of reporter constructs which test the effects of sequence elements from the control region of human mitochondrial DNA on expression in the nucleus, as assayed by transient expression in cultured human cells. The mitochondrial heavy-strand promoter (HSP) was unable to function as a promoter in nuclear DNA. Neither the HSP, nor the binding region for the mitochondrial transcription factor mtTF1 from the light-strand promoter, had any significant or systematic modulatory effects upon transcription from strong or weak RNA polymerase II (pol II) promoters, in three different human cell lines. The same finding held true regardless of orientation with respect to the start site of transcription. Similar results were obtained with a rho 0 derivative of one of these lines, indicating that mitochondrial promoter sequences in the nucleus cannot modulate transcription in response to altered mtDNA copy number. These results support the view that the nuclear and mitochondrial transcription systems in human cells are functionally independent, and do not communicate through factors recognizing shared sequence elements, as suggested by studies in yeast.
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PMID:Apparent functional independence of the mitochondrial and nuclear transcription systems in cultured human cells. 783 Jul 24

A diversity of promoter structures. It is evident that tremendous diversity exists between the modes of mitochondrial transcription initiation in the different eukaryotic kingdoms, at least in terms of promoter structures. Within vertebrates, a single promoter for each strand exists, which may be unidirectional or bidirectional. In fungi and plants, multiple promoters are found, and in each case, both the extent and the primary sequences of promoters are distinct. Promoter multiplicity in fungi, plants and trypanosomes reflects the larger genome size and scattering of genes relative to animals. However, the dual roles of certain promoters in transcription and replication, at least in yeast, raises the interesting question of how the relative amounts of RNA versus DNA synthesis are regulated, possibly via cis-elements downstream from the promoters. Mitochondrial RNA polymerases. With respect to mitochondrial RNA polymerases, characterization of human, mouse, Xenopus and yeast enzymes suggests a marked degree of conservation in their behavior and protein composition. In general, these systems consist of a relatively non-selective core enzyme, which itself is unable to recognize promoters, and at least one dissociable specificity factor, which confers selectivity to the core subunit. In most of these systems, components of the RNA polymerase have been shown to induce a conformational change in their respective promoters and have also been assigned the role of a primase in the replication of mtDNA. While studies of the yeast RNA polymerase have suggested it has both eubacterial (mtTFB) and bacteriophage (RPO41) origins, it is not yet clear whether these characteristics will be conserved in the mitochondrial RNA polymerases of all eukaryotes. mtTFA-mtTFB; conserved but dissimilar functions. With respect to transcription factors, mtTFA has been found in both vertebrates and yeast, and may be a ubiquitous protein in mitochondria. However, the divergence in non-HMG portions of the proteins, combined with differences in promoter structure, has apparently relegated mtTFA to alternative, or at least non-identical, physiological roles in vertebrates and fungi. The relative ease with which mtTFA can be purified (Fisher et al. 1991) suggests that, where present, it should be facile to detect. mtTFB may represent a eubacterial sigma factor adapted for interaction with the mitochondrial RNA polymerase.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Mitochondrial transcription initiation: promoter structures and RNA polymerases. 852 66

In previous studies the AZF1 gene has been identified as a second high-copy number suppressor for a special mutant of the gene for the mitochondrial core enzyme of RNA polymerase. The first high-copy number suppressor of this mutant turned out to be the specificity factor MTF1 for mitochondrial transcription. Up to now, the influence of AZF1 on mitochondrial transcription, its precise localization in the cell and the regulation of its expression has not been determined. The putative protein contains a long stretch of poly-asparagine amino acids and a typical zinc-finger domain for DNA binding. These characteristic structural features were used to create the abbreviation AZF1 (Asparagine-rich Zinc Finger protein). An initial computer analysis of the sequence gave no conclusive results for the presence of a mitochondrial import sequence or a typical nuclear-targeting sequence. A recent more-detailed analysis identified a possible nuclear localization signal in the middle of the protein. Disruption of the gene shows no effect on plates with glucose-rich medium or glycerol. In this report a specific polyclonal antibody against Azf1p was prepared and used in cell-fractionation experiments and in electron-microscopic studies. Both of these clearly demonstrate that the AZF1 protein is localized exclusively in the nucleus of the yeast cell. Northern analysis for the expression of the AZF1 messenger RNA under different growth conditions was therefore performed to obtain new insights into the regulation of this gene. Together with the respective protein-expression analysis these data demonstrate that Azf1p is preferentially synthezised in higher amounts under non-fermentable growth conditions. Over-expression of Azf1p in the yeast cell does not influence the expression level of the mitochondrial transcription factor Mtf1p, indicating that the influence of Azf1p on the suppression of the special mitochondrial RNA polymerase mutant is an indirect one. Subcellular investigation of the deletion mutant by electron microscopy identifies specific ultrastructural cell-division defects in comparison to the wild-type.
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PMID:Azf1p is a nuclear-localized zinc-finger protein that is preferentially expressed under non-fermentative growth conditions in Saccharomyces cerevisiae. 979 62

In the present work, the RNA polymerase activity of the human mitochondrial RNA polymerase mature protein (h-mtRPOLm) is shown, and its molecular activity calculated (2.1+/-0.9 min(-1)). An activity analysis of h-mtRPOLm and deleted versions of it has demonstrated that the entire recombinant protein is required for this activity. In addition, h-mtRPOLm alone or in presence of the known mitochondrial transcription factors (human mitochondrial transcription factor A and/or human mitochondrial transcription termination factor) is not able to initiate transcription from the specific human mitochondrial promoters pointing to the existence of a human mitochondrial transcription factor B-like protein.
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PMID:A study on the human mitochondrial RNA polymerase activity points to existence of a transcription factor B-like protein. 1151 53

Anticancer activity of cisplatin (cis-diamminedichloroplatinum) is believed to result from its interaction with DNA. The drug reacts with nucleophilic sites in DNA forming monoadducts as well as intra- and interstrand crosslinks. DNA-cisplatin adducts are specifically recognized by several proteins. They can be divided into two classes. One constitutes proteins which recognize DNA damage as an initial step of the nucleotide excision and mismatch repair pathways. The other class contains proteins stabilizing cellular DNA-protein and protein-protein complexes, including non-histone proteins from the HMG (high-mobility-group) family. They specifically recognize 1,2-interstrand d(GpG) and d(ApG) crosslinks of DNA-cisplatin adducts and inhibit their repair. Many HMG-domain proteins can function as transcription factors, e.g. UBF, an RNA polymerase I transcription factor, the mammalian testis-determining factor SRY and the human mitochondrial transcription factor mtTFA. Moreover, it seems that some proteins, which probably recognize DNA-cisplatin adducts non-specifically, e.g. actin and other nuclear matrix proteins, can disturb the structural and functional organization of the nucleus and whole cell. The formation of complexes between DNA and proteins in the presence of cisplatin and the changes in the cell architecture may account for the drug cytotoxicity.
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PMID:Recognition and repair of DNA-cisplatin adducts. 1242 29

The mitochondrial RNA polymerase (mtRNAP) from Saccharomyces cerevisiae (yeast) is composed of two nuclear encoded proteins, the core RNA polymerase (Rpo41) and the mitochondrial transcription factor (Mtf1). Although Rpo41 is strikingly similar to the single subunit RNAPs from the T7 and T3 bacteriophage (T7RNAP), the core mtRNAP requires Mtf1 for accurate transcription from a linear promoter-containing DNA template, while T7RNAP does not require any other additional factors for promoter selectivity. The fact that the mtRNAP requires an additional promoter utilization factor makes it an excellent model system for the analysis of the transitions that occur during transcription initiation. However, large-scale purification of the 153 kDa Rpo41 has only been reported from yeast cells, or as a recombinant from baculovirus, both sources requiring extensive purification with poor yields. We have developed a His-tagged Rpo41 expression construct suitable for rapid purification of large amounts of soluble Rpo41 from bacterial cells. Transcriptionally active forms of both wild type and point mutants of Rpo41 can be purified by a combination of batch ion exchange chromatography to remove nucleic acids and nickel affinity chromatography. An additional advantage of the isolation of Rpo41 from bacterial cells is the absence of its associated specificity factor Mtf1. This allows analysis of combinations of mutant forms of both components of the mtRNAP holoenzyme.
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PMID:Expression and purification of wild type and mutant forms of the yeast mitochondrial core RNA polymerase, Rpo41. 1503 75


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