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)

Angiosperms possess a small family of phage-type RNA polymerase genes that arose by gene duplication from an ancestral gene encoding the mitochondrial RNA polymerase. We have isolated and sequenced the genes and cDNAs encoding two phage-type RNA polymerases, PpRpoT1 and PpRpoT2, from the moss Physcomitrella patens. PpRpoT1 comprises 19 exons and 18 introns, PpRpoT2 contains two additional introns. The N-terminal transit peptides of both polymerases are shown to confer dual-targeting of green fluorescent protein fusions to mitochondria and plastids. In vitro translation of the cDNAs revealed initiation of translation at two in-frame AUG start codons. Translation from the first methionine gives rise to a plastid-targeted polymerase, whereas initiation from the second methionine results in exclusively mitochondrial-targeted protein. Thus, dual-targeting of Physcomitrella RpoT is caused by and might be regulated by multiple translational starts. In phylogenetic analyses, the Physcomitrella RpoT polymerases form a sister group to all other phage-type polymerases of land plants. The two genes result from a gene duplication event that occurred independently from the one which led to the organellar polymerases with mitochondrial or plastid targeting properties in angiosperms. Yet, according to their conserved exon-intron structures they are representatives of the molecular evolutionary line leading to the RpoT genes of higher land plants.
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PMID:Two RpoT genes of Physcomitrella patens encode phage-type RNA polymerases with dual targeting to mitochondria and plastids. 1206 4

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

Two classes of RNA polymerases transcribe RNA from promoters on DNA templates: promoter recognition-competent single polypeptides and multisubunit enzymes that require separable promoter recognition factors. Eukaryotic mitochondria utilize an unusual hybrid of these classes composed of a "core" RNA polymerase related to the single polypeptide enzymes plus a "specificity factor" necessary for promoter utilization. Using supercoiled or premelted templates, we have discovered that the yeast core mitochondrial RNA polymerase (Rpo41) has the intrinsic ability to initiate from promoters without its specificity factor (Mtf1). Rpo41 requires the mitochondrial promoter sequence (ATATAAGTA) for this activity. On premelted templates addition of Mtf1 actually inhibits the promoter selective activity of Rpo41. Mtf1 increases abortive relative to productive transcription by Rpo41, possibly by stabilizing the promoter complex and reducing escape into elongation. The requirement for Mtf1 on closed but not open templates indicates that Mtf1 facilitates melting but not recognition of promoters.
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PMID:Intrinsic promoter recognition by a "core" RNA polymerase. 1534 28

By analyzing ESTs that correspond to human POLRMT gene encoding mitochondrial RNA polymerase (mtRNAP) we revealed an alternatively spliced transcript. We confirmed the existence of the transcript that contain additional 225 nucleotides from proximal part of intron 1 by RT-PCR using RNA from HeLa cells. In mouse and rat there are similar alternative transcripts that contain entire intron 1 sequences. In addition, in mouse we revealed third transcript that contain extra exon derived from 142 bp of intron 2. The revealed alternative transcripts, in contrast to the mRNA encoding mtRNAP, specify N-terminally truncated protein lacking mitochondrial targeting signal. This protein has strictly nuclear localization and corresponds to nuclear RNA polymerase IV that we recently identified.
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PMID:[Alternative transcripts from POLRMT responsible for synthesis of nuclear RNA polymerase IV]. 1577 49

Transcription of eukaryotic genes is performed by three nuclear RNA polymerases, of which RNA polymerase II is thought to be solely responsible for the synthesis of messenger RNAs. Here we show that transcription of some mRNAs in humans and rodents is mediated by a previously unknown single-polypeptide nuclear RNA polymerase (spRNAP-IV). spRNAP-IV is expressed from an alternative transcript of the mitochondrial RNA polymerase gene (POLRMT). The spRNAP-IV lacks 262 amino-terminal amino acids of mitochondrial RNA polymerase, including the mitochondrial-targeting signal, and localizes to the nucleus. Transcription by spRNAP-IV is resistant to the RNA polymease II inhibitor alpha-amanitin but is sensitive to short interfering RNA specific for the POLRMT gene. The promoters for spRNAP-IV differ substantially from those used by RNA polymerase II, do not respond to transcriptional enhancers and contain a common functional sequence motif.
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PMID:Transcription of mammalian messenger RNAs by a nuclear RNA polymerase of mitochondrial origin. 2529 40

In the in vitro mitochondrial (mt) transcription initiation system with mt RNA polymerase fraction and mt lysate, the transcription initiation products were shown to be synthesized bidirectionally from the only H-strand-promoter (HSP)/L-strand-promoter region (LSP) of the mitochondrial D-loop genome segment. These transcription products ranged between >100 and >800 bp with the purified mitochondrial RNA polymerase fraction, but were larger (>2030-4000 bp) in size with the mitochondrial lysate in both human and mouse. In this brief report, an in vitro reconstituted mitochondrial transcription system purified by affinity chromatography (heparin-Sepharose) from mouse hypotetraploid letter Ehrlich ascites tumor cell mitochondria was shown to initiate transcription bidirectionally from the mitochondrial D-loop region (HSP/LSP), as evidenced by in vitro generated transcription products. The in vitro generated transcription products were separated by sequencing gel. But this in vitro reconstituted transcription system was not studied beyond the D-loop region. A 3D model of the enzyme RNA polymerase was docked with both ATP and CTP.
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PMID:3D model of RNA polymerase and bidirectional transcription. 1728 94

Understanding the details of how genetic information is expressed from the separate mitochondrial genome requires a detailed description of the properties of the mitochondrial RNA polymerase. This nuclear-encoded enzyme is necessary and sufficient for the transcription of all mitochondrially encoded genes. Mitochondria from yeast to humans use a single-polypeptide catalytic RNA polymerase related to enzymes from bacteriophage. They also require separable transcription factors necessary for initiation at promoter sequences on the mitochondrial DNA template. It has recently become possible to work with highly purified, recombinant forms of the mitochondrial RNA polymerase subunits from yeast. This chapter describes detailed protocols for working in vitro with this purified enzyme in transcription reactions. These assays are critical for elucidating the nature of a mitochondrial promoter and for understanding how the mitochondrial RNA polymerase recognizes these DNA sequences and selectively initiates the transcription cycle, resulting in discrete transcripts.
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PMID:In vitro analysis of the yeast mitochondrial RNA polymerase. 1831 27

Hybridization between populations can disrupt gene expression, frequently resulting in deleterious hybrid phenotypes. Reduced fitness in interpopulation hybrids of the marine copepod Tigriopus californicus has been traced to interactions between the nuclear and mitochondrial genomes. Here, we determine transcript levels of four to six genes involved in the mitochondrial oxidative phosphorylation pathway for a series of parental and inbred hybrid lines using RT-qPCR. Both nuclear and mitochondrial-encoded genes are included in the analysis. Although all genes studied are up-regulated under salinity stress, only expression of genes located on the mtDNA differed among lines. Because mitochondrial genes are transcribed by a dedicated RNA polymerase encoded in the nuclear genome, we compare transcript levels among hybrid lines with different combinations of mitochondrial RNA polymerase and mtDNA genotypes. Lines bearing certain mtDNA-mitochondrial RNA polymerase genotypic combinations show a diminished capacity to up-regulate mitochondrial genes in response to hypoosmotic stress. Effects on the transcriptional profile depend on the specific interpopulation cross and are correlated with viability effects. We hypothesize that disruption of the mitochondrial transcriptional system in F(2) hybrids may play a central role in hybrid breakdown.
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PMID:Genotype-dependent variation of mitochondrial transcriptional profiles in interpopulation hybrids. 1884 6

The mitochondrial RNA polymerase (mtRNAP) of Saccharomyces cerevisiae, consisting of a complex of Rpo41 and Mtf1, is homologous to the phage single polypeptide T7/T3 RNA polymerases. The yeast mtRNAP recognizes a conserved nonanucleotide sequence to initiate specific transcription. In this work, we have defined the region of the nonanucleotide that is melted by the mtRNAP using 2-aminopurine (2AP) fluorescence that is sensitive to changes in base stacking interactions. We show that mtRNAP spontaneously melts the promoter from -4 to +2 forming a bubble around the transcription start site at +1. The location and size of the DNA bubble in this open complex of the mtRNAP closely resembles that of the T7 RNA polymerase. We show that DNA melting requires the simultaneous presence of Rpo41 and Mtf1. Adding the initiating nucleotide ATP does not expand the size of the initially melted DNA, but the initiating nucleotide differentially affects base stacking interactions at -1 and -2. Thus, the promoter structure upstream of the transcription start site is slightly rearranged during early initiation from its structure in the pre-initiation stage. Unlike on the duplex promoter, Rpo41 alone was able to form a competent open complex on a pre-melted promoter. The results indicate that Rpo41 contains the elements for recognizing the melted promoter through interactions with the template strand. We propose that Mtf1 plays a role in base pair disruption during the early stages of open complex formation.
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PMID:Fluorescence mapping of the open complex of yeast mitochondrial RNA polymerase. 1911 3

Characterization of the basic transcription machinery of mammalian mitochondrial DNA has been greatly supported by the availability of pure recombinant mitochondrial RNA polymerase (mtRNAP) and accessory factors, which allowed to develop a reconstituted in vitro transcription system. This chapter outlines a general strategy that makes use of a minimal promoter-independent transcription assay to study mitochondrial transcription termination in animal systems. We used such a system to investigate the transcription termination properties of the sea urchin factor mtDBP, however, it is applicable to the study of transcription termination in a variety of organisms, provided that the pure mtRNAP and the transcription termination factor are available.The assay here described contains the recombinant proteins mtRNAP and mtDBP, both expressed in insect cells, and a template consisting of a 3'-tailed DNA construct bearing the sequence bound by mtDBP. Transcription by the RNA polymerase produces run-off and terminated molecules, the size of the latter being consistent with RNA chain arrest in correspondence of the mtDBP-DNA complex. Transcription termination is protein-dependent as addition of increasing amounts of mtDBP to the assay causes a decrease in the intensity of the run-off and the gradual appearance of short-terminated molecules. Furthermore, we report a method, based on pulse-chase experiments, which allows us to distinguish between the true termination and the pausing events.
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PMID:Methods for studying mitochondrial transcription termination with isolated components. 1951 72


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