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)

Histones have been both radiolabeled and density-labeled with amino acids in vivo to determine the dynamics of histone-DNA and histone-histone interactions at the replication fork and on active genes. Proteins were uniformly labeled and subsequently chased for three cell generations. During the chase period, H3,H4 tetramers dissociated from the H2A,H2B dimers to re-form nucleosomes with the corresponding nondense histones synthesized during the chase period. These data suggest that the prereplicative nucleosomes are dissolved during advancement of the replication fork with release of associated histones in the form of the H3,H4 tetramers and H2A,H2B dimers. Experiments that involve density labeling of cells in the presence of actinomycin D indicate that the dynamic exchange of H2A,H2B that has been previously described [Jackson, V. (1987) Biochemistry 26, 2315-2324] is partially dependent on RNA polymerase movement. These results provide indirect evidence that nucleosome dissolution occurs during transcription. When deposition during replication and transcription is inhibited by simultaneous treatment of cells with cytosine arabinoside and actinomycin D, the majority of the newly synthesized histones are unable to deposit into nucleosome structure. The low level of deposition that is observed has characteristics similar to the deposition of uH2A and uH2B, and it is proposed that conjugation of H2A and H2B by ubiquitin occurs when these proteins are in a free pool within the nucleus. The new H3,H4 tetramers and new H2A,H2B dimers when prevented from depositing are not stable. New and old H3 and H4 intermix to form hybrid tetramers, and a similar intermixing is observed for the H2A,H2B dimers. A model is presented to describe the dynamics of histone-DNA interactions during replication and transcription.
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PMID:In vivo studies on the dynamics of histone-DNA interaction: evidence for nucleosome dissolution during replication and transcription and a low level of dissolution independent of both. 169 79

Upon infection of animal cells by Sindbis virus, four nonstructural (ns) proteins, termed nsP1-4 in order from 5' to 3' in the genome, are produced by posttranslational cleavage of a polyprotein. nsP4 is believed to function as the viral RNA polymerase and is short-lived in infected cells. We show here that nsP4 produced in reticulocyte lysates is degraded by the N-end rule pathway, one ubiquitin-dependent proteolytic pathway. When the N-terminal residue of nsP4 is changed by mutagenesis, the metabolic stabilities of the mutant nsP4s follow the N-end rule, in that the half-life of nsP4 bearing different N-terminal residues decreases in the order Met greater than Ala greater than Tyr greater than or equal to Phe greater than Agr. Addition of dipeptides Tyr-Ala, Trp-Ala, or Phe-Ala to the translation mixture inhibits degradation of Tyr-nsP4 and Phe-nsP4, but not of Arg-nsP4. Conversely, dipeptides His-Ala, Arg-Ala, and Lys-Ala inhibit the degradation of Arg-nsP4 but not of Tyr-nsP4 or Phe-nsP4. We found that there is no lysine in the first 43 residues of nsP4 that is required for its degradation, indicating that a more distal lysine functions as the ubiquitin acceptor. Strict control of nsP4 concentration appears to be an important aspect of the virus life cycle, since the concentration of nsP4 in infected cells is regulated at three levels: translation of nsP4 requires read-through of an opal termination codon such that it is underproduced; differential processing by the virus-encoded proteinase results in temporal regulation of nsP4; and nsP4 itself is a short-lived protein degraded by the ubiquitin-dependent N-end rule pathway.
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PMID:Sindbis virus RNA polymerase is degraded by the N-end rule pathway. 192 57

Highly purified chicken progesterone receptor (cPR) is shown to stimulate RNA synthesis directly in an in vitro transcription assay. Stimulation of transcription by cPR requires the presence of progesterone response elements (PREs) in the template and can be specifically inhibited by addition of competitor oligonucleotides containing PREs. Binding of receptor to two PREs is cooperative and leads to synergistic (27-fold) stimulation of transcription. A purified fusion protein containing the DNA binding domain of cPR linked to yeast ubiquitin was produced in E. coli and also functions in the transcription assay. Using this in vitro transcription system, we demonstrate that hormone-free cPR activated by salt treatment induces transcription of a test gene in a hormone-independent manner. Finally, we present evidence that the progesterone receptor acts by facilitating the formation of a stable preinitiation complex at the target gene promoter and thus augments the initiation of transcription by RNA polymerase II.
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PMID:The progesterone receptor stimulates cell-free transcription by enhancing the formation of a stable preinitiation complex. 215 62

We have investigated the induction of known hsp (heat shock protein) RNA and other heat shock (HS) inducible transcripts in Chinese hamster cells by various stresses including DNA damaging agents. cDNA clones coding for at least 14 different HS-inducible transcripts were isolated. By DNA sequence analysis and homology with cDNA clones of other species, some of these cDNA clones were identified as coding for hsp27, hsp89 alpha, hsp89 beta, two different hsp70s, ubiquitin, and the HS-inducible RNA polymerase III transcript B2. In addition, hsp-related cDNA clones, hsp60 and four with hsp70 homology, were isolated which coded for transcripts which were not induced by HS or other stresses in two different Chinese hamster cell lines. After HS or treatment with the HS-mimetic agent ethanol, there was coordinate induction of all 14 transcripts. With severe HS treatments which produced substantial cytotoxicity, the increase in all transcripts except B2 RNA was delayed and, in some cases, suppressed. The only DNA damaging agent, which induced many HS-inducible transcripts, was high-dose methylmethane sulfonate (MMS). However, induction by MMS was not coordinate for all transcripts as it was for HS, and B2 RNA was not induced. hsp27 RNA induction differed from the others in several respects including induction by irradiation and other agents which produce high levels of DNA damage repaired by nucleotide excision repair. The implications of these findings in cellular events such as cytotoxicity, thermotolerance, and regulation of stress responses will be discussed.
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PMID:Induction of heat shock protein transcripts and B2 transcripts by various stresses in Chinese hamster cells. 254 Oct 7

In eukaryotic cells ubiquitin is synthesized as a polyubiquitin protein or as a protein fused at the carboxyl terminus to other polypeptides. An enzyme activity, ubiquitin protein peptidase, has been proposed to process these precursors by cleaving the peptide bond between adjoining ubiquitin molecules or between ubiquitin and the fused peptides. Using the cleavage of a 35S-labeled yeast ubiquitin protein fused to a synthetic 38-residue peptide obtained by in vivo metabolic labeling in Escherichia coli in an expression system based on the interaction of bacteriophage T7 RNA polymerase and its promoter, it is possible to detect a processing activity in soluble yeast extract. The specificity of the cleavage suggests this activity could be the in vivo processing activity for various ubiquitin precursor proteins in yeast cells. A similarly labeled ubiquitin protein fused to one cysteine residue was also utilized to detect an activity capable of removing a single cysteine residue from ubiquitin in a soluble extract. Employing assays based on the cleavage of labeled ubiquitin protein fusions, a ubiquitin protein peptidase activity from Saccharomyces cerevisiae was purified about 15,000-fold to yield a protein mixture consisting of only a few protein species. The major protein band which comigrated with the activities in in vitro assays has an apparent molecular weight of 29,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two other protein species, about 20,000 and 10,000 in molecular weight, also comigrated with the in vitro activities throughout the purification procedure. Though our most purified protein fraction was shown to cleave various artificial ubiquitin protein fusions under our experimental conditions, it cannot cleave a ubiquitin dimer protein, suggesting the existence of functionally distinct ubiquitin protein peptidases. Our experimental protocol for preparing various labeled ubiquitin protein precursors provides a means to explore various processing enzymes existing in cells. The same protocol may also be adapted to prepare substrates for the study of other specific protein processing enzymes.
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PMID:Purification of a ubiquitin protein peptidase from yeast with efficient in vitro assays. 255 55

General transcription factor SIII, a heterotrimer composed of 110-kDa (p110), 18-kDa (p18), and 15-kDa (p15) subunits, increases the catalytic rate of transcribing RNA polymerase II by suppressing transient pausing by polymerase at multiple sites on DNA templates. Here we report molecular cloning and biochemical characterization of the SIII p18 subunit, which is found to be a member of the ubiquitin homology (UbH) gene family and functions as a positive regulatory subunit of SIII. p18 is a 118-amino acid protein composed of an 84-residue N-terminal UbH domain fused to a 34-residue C-terminal tail. Mechanistic studies indicate that p18 activates SIII transcriptional activity above a basal level inherent in the SIII p110 and p15 subunits. Taken together, these findings establish a role for p18 in regulating the activity of the RNA polymerase II elongation complex, and they bring to light a function for a UbH domain protein in transcriptional regulation.
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PMID:Positive regulation of general transcription factor SIII by a tailed ubiquitin homolog. 763 63

The Elongin (SIII) complex activates elongation by mammalian RNA polymerase II by suppressing transient pausing of the polymerase at many sites within transcription units. Elongin is a heterotrimer composed of A, B, and C subunits of 110, 18, and 15 kilodaltons, respectively. Here, the mammalian Elongin A gene was isolated and expressed, and the Elongin (SIII) complex reconstituted with recombinant subunits. Elongin A is shown to function as the transcriptionally active component of Elongin (SIII) and Elongin B and C as regulatory subunits. Whereas Elongin C assembles with Elongin A to form an AC complex with increased specific activity, Elongin B, a member of the ubiquitin-homology gene family, appears to serve a chaperone-like function, facilitating assembly and enhancing stability of the Elongin (SIII) complex.
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PMID:Elongin (SIII): a multisubunit regulator of elongation by RNA polymerase II. 766 Jan 21

This article presents the development of a set of new expression vectors for overproduction of proteins in Escherichia coli. The vectors, pETUBI-ES1, 2 and 3, allow in-frame cloning of any sequence with the ubiquitin gene driven by the strong T7f10 promoter. Combination of the T7 expression system with ubiquitin fusion appears to have a synergistic effect on protein overproduction. Large amounts of stable RNA are produced by T7 RNA polymerase, and fusion of ubiquitin to the N-terminus of target proteins seems to confer more efficient translation, better folding or protection against proteolytic degradation. The ubiquitin part can be utilized for purification of the fusion protein, after which it can be easily removed from the fusion product by ubiquitin-specific proteases. The advantage of combining both systems is demonstrated by the synthesis of large quantities (up to 40-50% of the total protein) of the human ERCC1 protein that hitherto was refractory to overproduction in various other E. coli and yeast expression systems.
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PMID:Augmentation of protein production by a combination of the T7 RNA polymerase system and ubiquitin fusion: overproduction of the human DNA repair protein, ERCC1, as a ubiquitin fusion protein in Escherichia coli. 839 22

The SUG1 gene of Saccharomyces cerevisiae encodes a putative ATPase. Mutations in SUG1 were isolated as suppressors of a mutation in the transcriptional activation domain of GAL4. Sug1 was recently proposed to be a subunit of the RNA polymerase II holoenzyme and to mediate the association of transcriptional activators with holoenzyme. We show here that Sug1 is not a subunit of the holoenzyme, at least in its purified form, but of the 26S proteasome, a large complex of relative molecular-mass 2,000K that catalyses the ATP-dependent degradation of ubiquitin-protein conjugates. Sug1 co-purifies with the proteasome in both conventional and nickel-chelate affinity chromatography. Our observations account for the reduced ubiquitin-dependent proteolysis in sug1 mutants and suggest that the effects of sug1 mutations on transcription are indirect results of defective proteolysis.
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PMID:Identification of the gal4 suppressor Sug1 as a subunit of the yeast 26S proteasome. 862 1

The E3 ubiquitin-protein ligases play an important role in controlling substrate specificity of the ubiquitin proteolysis system. A biochemical approach was taken to identify substrates of Rsp5, an essential hect (homologous to E6-AP carboxyl terminus) E3 of Saccharomyces cerevisiae. We show here that Rsp5 binds and ubiquitinates the largest subunit of RNA polymerase II (Rpb1) in vitro. Stable complex formation between Rsp5 and Rpb1 was also detected in yeast cell extracts, and repression of RSP5 expression in vivo led to an elevated steady-state level of Rpb1. The amino-terminal domain of Rsp5 mediates binding to Rpb1, while the carboxyl-terminal domain of Rpb1, containing the heptapeptide repeats characteristic of polymerase II, is necessary and sufficient for binding to Rsp5. Fusion of the Rpb1 carboxyl-terminal domain to another protein also causes that protein to be ubiquitinated by Rsp5. These findings indicate that Rsp5 targets at least a subset of cellular Rpb1 molecules for ubiquitin-dependent degradation and may therefore play a role in regulating polymerase II activities. In addition, the results support a model for hect E3 function in which the amino-terminal domain mediates substrate binding, while the carboxyl-terminal hect domain catalyzes ubiquitination of bound substrates.
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PMID:The large subunit of RNA polymerase II is a substrate of the Rsp5 ubiquitin-protein ligase. 910 33


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