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)

When cells enter mitosis, RNA synthesis ceases. Yet the RNA polymerase I (pol I) transcription machinery involved in the production of pre-rRNA remains bound to the nucleolus organizing region (NOR), the chromosome site harboring the tandemly repeated rRNA genes. Here we examine whether rDNA transcription units are transiently blocked or "frozen" during mitosis. By using fluorescent in situ hybridization we were unable to detect nascent pre-rRNA chains on the NORs of mouse 3T3 and rat kangaroo PtK2 cells. Appropriate controls showed that our approach was sensitive enough to visualize, at the light microscopic level, individual transcriptionally active rRNA genes both in situ after experimental unfolding of nucleoli and in chromatin spreads ("Miller spreads"). Analysis of the cell cycle-dependent redistribution of transcript-associated components also revealed that most transcripts are released from the rDNA at mitosis. Upon disintegration of the nucleolus during mitosis, U3 small nucleolar RNA (snoRNA) and the nucleolar proteins fibrillarin and nucleolin became dispersed throughout the cytoplasm and were excluded from the NORs. Together, our data rule out the presence of "frozen Christmas-trees" at the mitotic NORs but are compatible with the view that inactive pol I remains on the rDNA. We propose that expression of the rRNA genes is regulated during mitosis at the level of transcription elongation, similarly to what is known for a number of genes transcribed by pol II. Such a mechanism may explain the decondensed state of the NOR chromatin and the immediate transcriptional reactivation of the rRNA genes following mitosis.
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PMID:A possible mechanism for the inhibition of ribosomal RNA gene transcription during mitosis. 773 Mar 96

Most DNA topoisomerase II (topo II) in cell-free extracts of 0-2-h old Drosophila embryos appears to be nonnuclear and remains in the supernatant after low-speed centrifugation (10,000 g). Virtually all of this apparently soluble topo II is particulate with a sedimentation coefficient of 67 S. Similar topo II-containing particles were detected in Drosophila Kc tissue culture cells, 16-19-h old embryos and extracts of progesterone-matured oocytes from Xenopus. Drosophila topo II-containing particles were insensitive to EDTA, Triton X-100 and DNase I, but could be disrupted by incubation with 0.3 M NaCl or RNase A. After either disruptive treatment, topo II sedimented at 9 S. topo II-containing particles were also sensitive to micrococcal nuclease. Results of chemical cross-linking corroborated those obtained by centrifugation. Immunoblot analyses demonstrated that topo II-containing particles lacked significant amounts of lamin, nuclear pore complex protein gp210, proliferating cell nuclear antigen, RNA polymerase II subunits, histones, coilin, and nucleolin. Northern blot analyses demonstrated that topo II-containing particles lacked U RNA. Thus, current data support the notion that nonnuclear Drosophila topo II-containing particles are composed largely of topo II and an unknown RNA molecule(s).
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PMID:An RNase-sensitive particle containing Drosophila melanogaster DNA topoisomerase II. 808 68

AMP nucleosidase (EC 3.2.2.4) from Escherichia coli and AMP deaminase (EC 3.5.4.6) from bakers' yeast are proposed to regulate cellular AMP levels under allosteric control of the activator ATP and the inhibitor, PO4. Both enzymes contain catalytic sites which bind AMP and regulatory sites which bind ATP. The deduced amino acid sequences of the proteins revealed only one region of homology in which six of eight amino acids are identical. A similar sequence is found in glyceraldehyde-3-phosphate dehydrogenase, phoE, ras proteins, RNA polymerase, K(+)-ATPase, nucleolin, and other proteins expected to have nucleotide or phosphate binding properties. In the crystal structure of glyceraldehyde-3-phosphate dehydrogenase, this sequence is part of the NAD(+)-binding site. The function of these amino acids was explored with a deletion mutant of AMP nucleosidase. The protein was over-produced in a pTZ construct using the AMP nucleosidase promoter which resulted in approximately 30% of the total protein as the desired enzyme. The mutation was characterized by DNA sequence analysis and by direct analysis of the peptides using high performance liquid chromatography-mass spectrometry. Deletion of amino acids 128-135, corresponding to DGSELTLD, produced an enzyme with a 20-fold decrease in Vmax but with smaller changes in substrate saturation kinetics, activation by MgATP, inhibition by inorganic phosphate, and inhibition by the tight-binding inhibitor, formycin 5-phosphate. The deletion mutant of AMP nucleosidase exhibits hysteresis in establishing a steady-state rate of product formation which is most pronounced in the absence of MgATP. These results establish that the sequence DGSELTLD in E. coli AMP nucleosidase is not required for binding of AMP, MgATP, or inorganic phosphate. However, the mutant enzyme has a structural defect related to the polymerization state which delays the onset of catalysis and decreases the catalytic efficiency.
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PMID:Mutagenic analysis of AMP nucleosidase from Escherichia coli. Deletion of a region similar to AMP deaminase and peptide characterization by mass spectrometry. 847 16

The nucleolar protein gar2, from the fission yeast Schizosaccharomyces pombe, is the functional homolog of NSR1 from Saccharomyces cerevisiae, and is structurally related to nucleolin from vertebrates. By immunocytochemistry at the electron microscope level, we show that gar2 co-localizes with RNA polymerase I and the gar1 protein along the dense fibrillar component of the nucleolus in a wild-type strain of S. pombe, suggesting that gar2 is involved in the transcription and/or in the early steps of maturation of the ribosomal RNAs. Since the effects of disruption of the gar2+ gene might also shed light on the role of the gar2 protein, we analyzed the ultrastructure of the nucleolus of a gar2-disruption mutant. The nucleolus of the gar2- mutant is dramatically reorganized when compared with that of the wild-type gar2+ strain: a truncated protein containing the NH2-terminus of the gar2 protein is accumulated in an unusual nucleolar "dense body". Our results also suggest that the NH2-terminus might be sufficient for nucleolar localization via interaction with specific nucleolar components and support the hypothesis that gar2 in wild-type S. pombe interacts with nascent pre-rRNA via its two RNA-binding domains in combination with the glycine/arginine-rich domain. We also report that disruption of the gar2+ gene results in a mutant that is defective in cytokinesis and nuclear division.
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PMID:Ultrastructural changes in the Schizosaccharomyces pombe nucleolus following the disruption of the gar2+ gene, which encodes a nucleolar protein structurally related to nucleolin. 921 82

We examined the distribution of the silver-stainable phosphoprotein, pp135, within Ehrlich tumor and HEp-2 cells by a postembedding Lowicryl immunogold labeling procedure. Identical labeling patterns were obtained in both cell types. During interphase, gold particles were found not only over the dense fibrillar component but were also evident over the fibrillar centers of nucleoli. By contrast, the granular component did not display any significant label. When rRNA synthesis was inhibited by actinomycin D, the same labeling was observed in segregated nucleoli; both fibrillar components were labeled. Aside from the nucleolar labeling, label was also consistently present in coiled bodies. During metaphase, label was visualized in silver-stainable material of the nucleolus organizing regions. It thus appears that, unlike the two major silver-stained proteins, nucleolin/C23 and B23, pp135 remains located in all major silver-stainable structures during the whole cell cycle. This finding strongly suggests that pp135 could be the component responsible for in situ silver staining. On the other hand, the maintenance of pp135 in the fibrillar centers throughout the cell cycle, like RNA polymerase I, upstream binding factor, and DNA topoisomerase I, suggests that pp135 could be a component involved in transcription of the rRNA genes.
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PMID:The phosphoprotein pp135 is an essential constituent of the fibrillar components of nucleoli and of coiled bodies. 972 Sep 89

During the early development of Xenopus laevis, we followed in individual nuclei the formation of a nucleolus by examining simultaneously its structural organization and its transcriptional competence. Three distinct situations were encountered with different frequencies during development. During the first period of general transcriptional quiescence, the transcription factor UBF of maternal origin, was present in most nuclei at the ribosomal gene loci. In contrast, fibrillarin, a major protein of the processing machinery, was found in multiple prenucleolar bodies (PNBs) whereas nucleolin was dispersed largely in the nucleoplasm. During the second period, for most nuclei these PNBs had fused into two domains where nucleolin concentrated, generating a structure with most features expected from a transcriptionally competent nucleolus. However, RNA polymerase I-dependent transcription was not detected using run-on in situ assays whereas unprocessed ribosomal RNAs were observed. These RNAs were found to derive from a maternal pool. Later, during a third period, an increasing fraction of the nuclei presented RNA polymerase I-dependent transcription. Thus, the structural organization of the nucleolus preceded its transcriptional competence. We conclude that during the early development of X. laevis, the organization of a defined nucleolar structure, is not associated with the transcription process per se but rather with the presence of unprocessed ribosomal RNAs.
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PMID:Presence of pre-rRNAs before activation of polymerase I transcription in the building process of nucleoli during early development of Xenopus laevis. 973 79

Nopp140 is thought to shuttle between nucleolus and cytoplasm. However, the predominant nucleolar localization of Nopp140 homologues from different species suggests that Nopp140 is also involved in events occurring within the nucleolus. In this study, we demonstrated that the largest subunit of RNA polymerase I, RPA194, was coimmunoprecipitated with the human Nopp140 (hNopp140). Such an interaction is mediated through amino acids 204 to 382 of hNopp140. By double immunofluorescence, hNopp140 was colocalized with RNA polymerase I at the rDNA (rRNA genes) transcription active foci in the nucleolus. These results suggest that Nopp140 can interact with RNA polymerase I in vivo. Transfected cells expressing the amino-terminal half of hNopp140, hNopp140N382 (amino acids 1 to 382), displayed altered nucleoli with crescent-shaped structures. This phenotype is reminiscent of the segregated nucleoli induced by actinomycin D treatment, which is known to inhibit rRNA synthesis. Consistently, the hNopp140N382 protein mislocalized the endogenous RNA polymerase I and shut off cellular rRNA gene transcription as revealed by an in situ run-on assay. These dominant negative effects of the mutant hNopp140N382 suggest that Nopp140 plays an essential role in rDNA transcription. Interestingly, ectopic expression of hNopp140 to a very high level caused the formation of a transcriptionally inactive spherical structure occupying the entire nucleolar area which trapped the RNA polymerase I, fibrillarin, and hNopp140 but excluded the nucleolin. The mislocalizations of these nucleolar proteins after hNopp140 overexpression imply that Nopp140 may also play roles in maintenance of nucleolar integrity.
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PMID:Human Nopp140, which interacts with RNA polymerase I: implications for rRNA gene transcription and nucleolar structural organization. 1056 78

Ribosome-inactivating proteins (RIPs) are RNA-N-glycosidases widely present in plants that depurinate RNA in ribosomes at a specific universally conserved position, A4324, in the rat 28S rRNA. A small group of RIPs (cofactor-dependent RIPs) require ATP and tRNA to reach maximal activity on isolated ribosomes. Among cofactor-dependent RIPs, gelonin is specifically and uniquely stimulated by tRNA(Trp). The active species are avian (chicken) and mammalian (beef, rat, and rabbit) tRNA(Trp), whereas yeast tRNA(Trp) is completely devoid of stimulating activity. In the present article, bovine and yeast tRNA(Trp) with unmodified bases were prepared by assembly of the corresponding genes from synthetic oligonucleotides followed by PCR and T7 RNA polymerase transcription of the amplified products. The two synthetic tRNAs were fully active (bovine) or inactive (yeast) as the wild-type tRNAs. Construction of chimeric tRNA(Trp) transcripts identified the following bovine nucleotides as recognition elements for gelonin-stimulating activity: G26 and bp G12-C23 in the D arm and G57, A59, and bp G51-C63 and U52-A62 in the T arm. Among single-stranded nucleotides, A59 has a prominent role, but full expression of the gelonin-stimulating activity requires an extensive cooperation between nucleotides in both arms.
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PMID:Identity elements in bovine tRNA(Trp) required for the specific stimulation of gelonin, a plant ribosome-inactivating protein. 1057 26

AgNOR proteins are a set of argyrophilic nucleolar proteins that accumulate in highly proliferating cells whereas their expression is very low in non-proliferating cells. Some of these proteins remain associated with the nucleolar organizer regions (NORs) during mitosis. In situ, the expression of AgNOR proteins is measured globally by quantification of the level of silver staining using morphometry and image analysis. To go deeper into the understanding of the relationship between the cell cycle and quantity of AgNOR proteins, it was necessary to determine the phases of cell cycle during which expression of AgNOR varies and what are the most variable proteins in each phase. To answer these questions, we set up the protocol permitting to detect and quantify AgNOR proteins on protein samples electrophoresed and transferred onto nitrocellulose membranes. This approach makes it possible to quantitatively evaluate individual AgNOR proteins and identify them, using nucleolar, nuclear and whole interphasic cell extracts, and chromosome-associated protein extracts. By this means, we identified nucleolin and protein B23 as the two major AgNOR proteins in the nucleolus during interphase and subunits of RNA polymerase I and transcription factor UBF as AgNOR proteins remaining associated with NORs during mitosis. We also observed that the increase in the level of nucleolin and protein B23 in rat liver seems to be linked with the cell cycle and not exclusively with stimulation of ribosomal gene (rDNA) transcription. Similarly in synchronized cells, the amount of nucleolin rapidly increases when cells enter the S phase (1.6-fold of the value of serum-deprived cells at 9 h, and 2.35-fold at 12 h after refeeding). The amount of protein B23 exhibits a lower and progressive increase with a maximum when the percentage of cells in G2 phase increased, i.e. after 24 h of cell cycle stimulation. We consider that the amount of AgNOR proteins can be a marker of proliferation, because this amount is related to cell cycle phases, schematically low for G1 phase and high for S-G2 phase. Thus, it is a measure of the relative proportion of cells in each phase, and consequently of the timing of each phase. The higher value indicates that the major part of the cells are in the S-G2 phase and correlatively few are in the G1 phase, and this characterizes a rapid cell cycle.
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PMID:The AgNOR proteins: qualitative and quantitative changes during the cell cycle. 1058 57

The c-Myc oncoprotein and its dimerization partner Max bind the DNA core consensus sequence CACGTG (E-box) and activate gene transcription. However, the low levels of induction have hindered the identification of novel Myc target genes by differential screening techniques. Here, we describe a computer-based pre-selection of candidate Myc/Max target genes, based on two restrictive criteria: an extended E-box consensus sequence for Myc/Max binding and the occurrence of this sequence within a potential genomic CpG island. Candidate genes selected by these criteria were evaluated experimentally for their response to Myc. Two Myc target genes are characterized here in detail. These encode nucleolin, an abundant nucleolar protein, and BN51, a co-factor of RNA polymerase III. Myc activates transcription of both genes via E-boxes located in their first introns, as seen for several well-characterized Myc targets. For both genes, mutation of the E-boxes abolishes transcriptional activation by Myc as well as repression by Mad1. In addition, the BN51 promoter is selectively activated by Myc and not by USF, another E-box-binding factor. Both nucleolin and BN51 are implicated in the maturation of ribosomal RNAs, albeit in different ways. We propose that Myc, via regulation of these and probably many other transcriptional targets, may be an important regulator of ribosome biogenesis.
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PMID:Myc induces the nucleolin and BN51 genes: possible implications in ribosome biogenesis. 1060 42


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