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)

A mutation in RPB5 (rpb5-9), an essential RNA polymerase subunit assembled into RNA polymerases I, II, and III, revealed a role for this subunit in transcriptional activation. Activation by GAL4-VP16 was impaired upon in vitro transcription with mutant whole-cell extracts. In vivo experiments using inducible reporter plasmids and Northern analysis support the in vitro data and demonstrate that RPB5 influences activation at some, but not all, promoters. Remarkably, this mutation maps to a conserved region of human RPB5 implicated by others to play a role in activation. Chimeric human-yeast RPB5 containing this conserved region now can function in place of its yeast counterpart. The defects noted with rpb5-9 are similar to those seen in truncation mutants of the RPB1-carboxyl terminal domain (CTD). We demonstrate that RPB5 and the RPB1-CTD have overlapping roles in activation because the double mutant is synthetically lethal and has exacerbated activation defects at the GAL1/10 promoter. These studies demonstrate that there are multiple activation targets in RNA polymerase II and that RPB5 and the CTD have similar roles in activation.
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PMID:RNA polymerase subunit RPB5 plays a role in transcriptional activation. 986 Sep 60

We have determined complete gene sequences encoding the largest subunit of the RNA polymerase II (RBP1) from two Microsporidia, Vairimorpha necatrix and Nosema locustae. Phylogenetic analyses of these and other RPB1 sequences strongly support the notion that Microsporidia are not early-diverging eukaryotes but instead are specifically related to Fungi. Our reexamination of elongation factors EF-1alpha and EF-2 sequence data that had previously been taken as support for an early (Archezoan) divergence of these amitochondriate protists show such support to be weak and likely caused by artifacts in phylogenetic analyses. These EF data sets are, in fact, not inconsistent with a Microsporidia + Fungi relationship. In addition, we show that none of these proteins strongly support a deep divergence of Parabasalia and Metamonada, the other amitochondriate protist groups currently thought to compose early branches. Thus, the phylogenetic placement among eukaryotes for these protist taxa is in need of further critical examination.
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PMID:Microsporidia are related to Fungi: evidence from the largest subunit of RNA polymerase II and other proteins. 989 76

The C-terminal domain (CTD) of the largest subunit (RPB1) of eukaryotic RNA polymerase II is essential for pol II function and has been shown to play a number of important roles in the mRNA transcription cycle. The CTD is composed of a tandemly repeated heptapeptide that is conserved in yeast, animals, plants and several protistan organisms. Some eukaryotes, however, have what appear to be degenerate or deviant CTD regions, and others have no CTD at all. The functional and evolutionary implications of this variation among RPB1 C-termini is largely unexplored. We have transformed yeast cells with a construct consisting of the yeast RPB1 gene with 25 heptads from the primitive protist Mastigamoeba invertens in place of the wild-type CTD. The Mastigamoeba heptads differ from the canonical CTD by the invariable presence of alanines in place of threonines at position 4, and in place of serines at position 7 of each heptad. Despite this double substitution, mutants are viable even under conditions of temperature and nutrient stress. These results provide new insights into the relative functional importance of several of the conserved CTD residues, and indicate that in vivo expression of evolutionary variants in yeast can provide important clues for understanding the origin, evolution and function of the pol II CTD.
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PMID:Evolutionary complementation for polymerase II CTD function. 1062 Jul 75

The subunits of Saccharomyces cerevisiae RNA polymerase II (RNAP II) in proximity to the DNA during transcription elongation have been identified by photoaffinity cross-linking. In the absence of transcription factors, RNAP II will transcribe a double-stranded DNA fragment containing a 3'-extension of deoxycytidines, a "tailed template". We designed a DNA template allowing the RNAP to transcribe 76 bases before it was stalled by omission of CTP in the transcription reaction. This stall site oriented the RNAP on the DNA template and allowed us to map the RNAP subunits along the DNA. The DNA analogue 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUTP (N(3)RdUTP) [Bartholomew, B., Kassavetis, G. A., Braun, B. R., and Geiduschek, E. P. (1990) EMBO J. 9, 2197-205] was synthesized and enzymatically incorporated into the DNA at specified positions upstream or downstream of the stall site, in either the template or nontemplate strand of the DNA. Radioactive nucleotides were positioned beside the photoactivatable nucleotides, and cross-linking by brief ultraviolet irradiation transferred the radioactive tag from the DNA onto the RNAP subunits. In addition to N(3)RdUTP, which has a photoreactive azido group 9 A from the uridine base, we used the photoaffinity cross-linker 5N(3)dUTP with an azido group directly on the uridine ring to identify the RNAP II subunits closest to the DNA at positions where multiple subunits cross-linked. In cross-linking reactions dependent on transcription, RPB1, RPB2, and RPB5 were cross-linked with N(3)RdUTP. With 5N(3)dUTP, only RPB1 and RPB2 were cross-linked. Under certain circumstances, RPB3, RPB4, and RPB7 were cross-linked. From the information obtained in this topological study, we developed a model of yeast RNAP II in a transcription elongation complex.
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PMID:Topology of yeast RNA polymerase II subunits in transcription elongation complexes studied by photoaffinity cross-linking. 1106 78

Bacterial DNA-dependent RNA polymerase (RNAP) has subunit composition beta'betaalpha(I)alpha(II)omega. The role of omega has been unclear. We show that omega is homologous in sequence and structure to RPB6, an essential subunit shared in eukaryotic RNAP I, II, and III. In Escherichia coli, overproduction of omega suppresses the assembly defect caused by substitution of residue 1362 of the largest subunit of RNAP, beta'. In yeast, overproduction of RPB6 suppresses the assembly defect caused by the equivalent substitution in the largest subunit of RNAP II, RPB1. High-resolution structural analysis of the omega-beta' interface in bacterial RNAP, and comparison with the RPB6-RPB1 interface in yeast RNAP II, confirms the structural relationship and suggests a "latching" mechanism for the role of omega and RPB6 in promoting RNAP assembly.
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PMID:Bacterial RNA polymerase subunit omega and eukaryotic RNA polymerase subunit RPB6 are sequence, structural, and functional homologs and promote RNA polymerase assembly. 1115 66

Molecular data have proved useful as an alternative to morphological data in showing the relationships of genera within the phylum Microsporidia, but until now have been available only for ribosomal genes. In previous studies protein-coding genes of microsporidia have been used only to assess their position in the evolution of eukaryotes. For the first time we report on the use of a protein-coding gene, the A-G region of the largest subunit of RNA polymerase II (RPB1) from 14 mainly polysporous species, to generate an alternative phylogeny for microsporidia. Using the amino acid sequences, the genera and species fell into the same main groupings as had been obtained with 16S rDNA sequences, but the RPB1 data provided better resolution within these groups. The results supported the pairings of Trachipleistophora hominis with Vavraia culicis and Pleistophora hippoglossoideos with Pleistophora typicalis. They also confirmed that the genus Pleistophora is not monophyletic and that it will be necessary to transfer Pleistophora ovariae and Pleistophora mirandellae into one or more other genera, as has already been effected for Pleistophora anguillarum.
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PMID:Relationships of microsporidian genera, with emphasis on the polysporous genera, revealed by sequences of the largest subunit of RNA polymerase II (RPB1). 1124 86

Transcriptional activity of RNA polymerase II is modulated during the cell cycle. We previously identified a temperature-sensitive mutation in the largest (catalytic) subunit of RNA polymerase II (RPB1) that causes cell cycle arrest and genome instability. We now characterize a different cell line that has a temperature-sensitive defect in cell cycle progression, and find that it also has a mutation in RPB1. The temperature-sensitive mutant, tsAF8, of the Syrian hamster cell line, BHK21, arrests at the non-permissive temperature in the mid-G(1) phase. We show that RPB1 in tsAF8--which is found exclusively in the nucleus at the permissive temperature--is also found in the cytoplasm at the non-permissive temperature. Comparison of the DNA sequences of the RPB1 gene in the wild-type and mutant shows the mutant phenotype results from a (hemizygous) C-to-A variation at nucleotide 944 in one RPB1 allele; this gives rise to an ala-to-asp substitution at residue 315 in the protein. Aligning the amino acid sequences from various species reveals that ala(315) is highly conserved in eukaryotes.
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PMID:A mutation in the largest (catalytic) subunit of RNA polymerase II and its relation to the arrest of the cell cycle in G(1) phase. 1167 99

Transcription of protein-coding genes by RNA polymerase II (pol II) is a highly coordinated process that requires the stepwise association of distinct protein complexes with the C-terminal domain (CTD) of Rpbl, the largest subunit of RNA pol II. Interaction of these complexes with the CTD might be subject to regulation by proteins such as Ess1 and Rsp5. Ess1, a prolyl-isomerase, binds the CTD and is thought to play a positive role in pol II transcription by generating conformational isomers of the CTD. Rsp5, a ubiquitin ligase, binds the CTD and is thought to play a negative role in transcription by mediating Rpbl ubiquitination and degradation. In this paper, we demonstrate that ESS1 and RSP5 interact genetically and that these interactions occur via RPBI. We show that over-expression of RSP5 enhances the growth defect of ess1ts cells and this effect is reversed by introducing extra copies of RPB1. Over-expression of RSP5 also mimics the sensitivity of ess1ts mutant cells to the toxicity of plasmids carrying dominant-negative CTD mutations, whereas mutations in RSP5 suppress this effect. Using a modified two-hybrid assay, we also demonstrate that Essl and Rsp5 compete directly for binding to the CTD. The results suggest a model in which Essl and Rsp5 act opposingly on pol II function to control the level of pol II available for transcription.
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PMID:Genetic interactions between the ESS1 prolyl-isomerase and the RSP5 ubiquitin ligase reveal opposing effects on RNA polymerase II function. 1179 43

In recent years a great deal of biochemical and genetic research has focused on the C-terminal domain (CTD) of the largest subunit (RPB1) of DNA-dependent RNA polymerase II. This strongly conserved domain of tandemly repeated heptapeptides has been linked functionally to important steps in the initiation and processing of mRNA transcripts in both animals and fungi. Although they are absolutely required for viability in these organisms, C-terminal tandem repeats do not occur in RPB1 sequences from diverse eukaryotic taxa. Here we present phylogenetic analyses of RPB1 sequences showing that canonical CTD heptads are strongly conserved in only a subset of eukaryotic groups, all apparently descended from a single common ancestor. Moreover, eukaryotic groups in which the most complex patterns of ontogenetic development occur are descended from this CTD-containing ancestor. Consistent with the results of genetic and biochemical investigations of CTD function, these analyses suggest that the enhanced control over RNA polymerase II transcription conveyed by acquired CTD/protein interactions was an important step in the evolution of intricate patterns of gene expression that are a hallmark of large, developmentally complex eukaryotic organisms.
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PMID:Evolution of the RNA polymerase II C-terminal domain. 1197 39

Through the use of photobleaching techniques, we examined the dynamic interaction of three members of the transcription apparatus with a target promoter in living cells. The glucocorticoid receptor (GR) interacting protein 1 (GRIP-1) exhibits a half maximal time for fluorescent recovery (tau(R)) of 5 s, reflecting the same rapid exchange as observed for GR. In contrast, the large subunit (RPB1) of RNA polymerase II (pol II) required 13 min for complete fluorescence recovery, consistent with its function as a processive enzyme. We also observe a complex induction profile for the kinetics of GR-stimulated transcription. Our results indicate that GR and GRIP-1 as components of the activating complex are in a dynamic equilibrium with the promoter, and must return to the template many times during the course of transcriptional activation.
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PMID:Dynamic behavior of transcription factors on a natural promoter in living cells. 1244 72


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