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On the phosphorylation of yeast RNA polymerases A and B 总被引:8,自引:0,他引:8
In exponentially growing cells, RNA polymerase B is exclusively form BI enzyme with several phosphorylated subunits: B220, B23 and possibly B44.5. In RNA polymerase A an average of fifteen phosphate groups are distributed on the five phosphorylated subunits: A190 (6), A43 (4), A34.5 (2), A23 (1-2) and A19 (1-2). Phosphorylation of enzyme A by a yeast protein kinase in vitro adds less than 1 mol phosphate/mol enzyme but occurs essentially at the physiological sites, as shown by a comparison of the peptide patterns obtained by limited proteolysis of subunits 32P-labelled in vivo and in vitro. No evidence was found in favor of a modulation of RNA polymerase activity in vitro or in vivo via phosphorylation. 相似文献
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Immunological studies of RNA polymerase II using antibodies to subunits of Drosophila and wheat germ enzyme 总被引:22,自引:0,他引:22
We induced goat antibodies to Drosophila RNA polymerase II and rabbit antibodies to the isolated 215,000-dalton and 140,000-dalton polymerase II subunits (P215 and P140, respectively). Similarly, we induced rabbit antibodies to wheat germ RNA polymerase II and to the 220,000-dalton subunit and 140,000-dalton subunit (P220 and P140, respectively). Anti-polymerase antibodies precipitated the homologous native enzyme and inhibited its activity in vitro, while several of the anti-subunit sera did neither. The anti-Drosophila P215 serum specifically labeled RNA polymerase II fixed in situ on polytene chromosomes. We reacted the antibodies with polymerase subunits separated by sodium dodecyl sulfate gel electrophoresis and electrophoretically transferred to nitrocellulose ("protein blotting"). Each antibody to whole polymerase reacted with multiple subunits, while the anti-subunit sera each reacted specifically with the subunit employed as immunogen. The anti-subunit sera also cross-reacted with the analogous subunit from several heterologous polymerases II (from yeast, wheat germ, Drosophila, and calf thymus), demonstrating shared subunit-specific determinants in polymerase II from widely divergent organisms. The anti-polymerase sera also showed cross-reactivity with subunits of heterologous enzymes, but only in one case did the cross-reactivity involve subunits other than the two largest ones. Specifically, the goat anti-Drosophila polymerase serum displayed easily detectable cross-reactivity with four low molecular weight subunits of calf thymus polymerase II, providing a unique demonstration of antigenic relatedness of small RNA polymerase II subunits from different higher eukaryotes. 相似文献
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B Bréant J Huet A Sentenac P Fromageot 《The Journal of biological chemistry》1983,258(19):11968-11973
Specific antibodies directed against each polypeptide component of yeast RNA polymerases A or B were prepared and their affinity spectrum determined by protein blot immunodetection. The majority of enzyme A or B subunits were specifically recognized by their respective antiserum. A direct correspondence was established between the polypeptides immunologically related in the three forms of RNA polymerases A, B, and C by reacting the different antibodies with enzymes subunits transferred to a nitrocellulose membrane. Subunit-specific antibodies and antibodies to native enzymes A and B were used to probe the activity of RNA polymerases A, B, and C. Based on DNA protection experiments, the largest subunit of enzymes A and B as well as the common subunit ABC23 appear to be involved in DNA binding. 相似文献
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Localization of yeast RNA polymerase I core subunits by immunoelectron microscopy. 总被引:1,自引:1,他引:0
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C Klinger J Huet D Song G Petersen M Riva E K Bautz A Sentenac P Oudet P Schultz 《The EMBO journal》1996,15(17):4643-4653
Immunoelectron microscopy was used to determine the spatial organization of the yeast RNA polymerase I core subunits on a three-dimensional model of the enzyme. Images of antibody-labeled enzymes were compared with the native enzyme to determine the localization of the antibody binding site on the surface of the model. Monoclonal antibodies were used as probes to identify the two largest subunits homologous to the bacterial beta and beta' subunits. The epitopes for the two monoclonal antibodies were mapped using subunit-specific phage display libraries, thus allowing a direct correlation of the structural data with functional information on conserved sequence elements. An epitope close to conserved region C of the beta-like subunit is located at the base of the finger-like domain, whereas a sequence between conserved regions C and D of the beta'-like subunit is located in the apical region of the enzyme. Polyclonal antibodies outlined the alpha-like subunit AC40 and subunit AC19 which were found co-localized also in the apical region of the enzyme. The spatial location of the subunits is correlated with their biological activity and the inhibitory effect of the antibodies. 相似文献
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Conservation of a DNA-binding site in the largest subunit of eukaryotic RNA polymerase II 总被引:10,自引:0,他引:10
Using a monoclonal antibody to a DNA-binding site of calf RNA polymerase II, we found that this site occurs on the largest subunit and is structurally similar in RNA polymerase II of widely divergent eukaryotes. In immuno-blotting of electrophoretically separated subunits, the monoclonal antibody recognized a determinant on the largest polypeptide of all RNA eukaryotic polymerase II forms tested, with a preference for the IIA enzyme subunit of 215 X 10(3) Mr over the partially proteolyzed 180 X 10(3) Mr form. This site is conserved on human, chicken, Drosophila, wheat germ and yeast RNA polymerase II, all of which reacted strongly with the monoclonal antibody. These results contrasted with those obtained with polyclonal antibodies to non-functional determinants of the calf enzyme. The reactivity of the polyclonal antibody with eukaryotic RNA polymerase II steadily decreased with increasing evolutionary distance from the original antigen; the yeast enzyme showed no cross-reactivity. These results suggest that a basic functional feature of eukaryotic RNA polymerase II has been strongly conserved and support the view that divergence of RNA polymerase II has taken place mainly in other, perhaps regulatory, sites of the enzyme. 相似文献
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RNA polymerases B and C are more closely related to each other than to RNA polymerase A 总被引:20,自引:0,他引:20
Amino acid sequence comparison of the largest subunit of the three forms of yeast nuclear RNA polymerase disclosed six major conserved regions that are partly retained in the cognate subunits from bacteria, viral, and insect enzymes (Mémet, S., Gouy, M., Marck, C., Sentenac, A., and Buhler, J.-M. (1988) J. Biol. Chem. 263, 2830-2839). Within these conserved domains, the high sequence similarity of B220 and C160 subunits (52% identity) sets them apart from yeast enzyme A subunit A190. Parsimony analysis at the gene and protein levels suggests the existence of a transient ancestor to eukaryotic RNA polymerases B and C. These results are discussed in the light of the recent finding of class C genes containing RNA polymerase B promoter elements. 相似文献
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Monoclonal antibodies directed against mammalian RNA polymerase I. Identification of the catalytic center 总被引:2,自引:0,他引:2
K M Rose K A Maguire J N Wurpel D A Stetler E D Márquez 《The Journal of biological chemistry》1983,258(21):12976-12981
Mouse myeloma cells were fused with splenocytes from a mouse that had been immunized with RNA polymerase I purified from a rat hepatoma. Hybridoma cells were selected and colonies secreting antibodies directed against the enzyme were detected by analysis of cell culture supernatants in a solid phase radioimmunoassay. Two of these cell lines were grown on a larger scale and the interaction between the immunoglobulins obtained from them and RNA polymerase I was studied in detail. Antibodies from both of the hybridoma cell lines were able to inhibit DNA-dependent RNA synthesis catalyzed by RNA polymerases I and III, but not that catalyzed by polymerase II. The antibodies were also capable of reducing the RNA chain elongation reaction catalyzed either by RNA polymerase I associated with isolated nucleoli or by enzyme preinitiated in vitro on calf thymus DNA. Inhibition of RNA polymerase I activity by the monoclonal antibodies was inversely related to the nucleotide concentration. In contrast, the DNA concentration had relatively little effect on inhibition by the antibodies. Analysis of immune complex formation between the antibodies and isolated individual enzyme subunits demonstrated that the monoclonal antibodies were directed against the largest (Mr = 190,000) polypeptide of the polymerase I. These data indicate that the largest subunit of RNA polymerase I contains an immunological determinant in common with RNA polymerase III and suggest that the polymerase I polypeptide of Mr = 190,000 contains a catalytic center involved in RNA chain elongation. 相似文献
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Zinc-binding subunits of yeast RNA polymerases 总被引:19,自引:0,他引:19
The zinc-binding subunits of yeast RNA polymerase A(I) and B(II) have been identified by a zinc-blotting technique. The two largest subunits of each enzyme (A190, A135, B220, and B150), as well as A12.2, A10, B44.5, B12.6, and B10, bind 65Zn(II). Predicted zinc-binding motifs have been noted in the NH2-terminal part of B220 and the COOH-terminal region of B150 subunits. Subdomains encompassing these motifs have been overproduced as MalE-fusion proteins and shown to retain zinc binding activity. Site-directed mutagenesis in the predicted metal-binding domain of B150 demonstrated its role in zinc binding. Mutations of cysteine residues C1163, C1166, C1182, and C1185 affected 65Zn2+ binding in vitro and caused a lethal or thermosensitive phenotype for growth. The ability to bind zinc is not sufficient for function since mutations in vicinal residues not affecting zinc binding were either lethal or thermosensitive. The role of zinc in RNA polymerase structure and function is discussed in the light of the present results. 相似文献
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Angela Krämer Regina Haars Rainer Kabisch Hans Will Friedlinde A. Bautz Ekkehard K. F. Bautz 《Molecular & general genetics : MGG》1980,180(1):193-199
Summary Monoclonal antibodies were raised against purified RNA polymerase II (or B) from Drosophila melanogaster. The antibody produced by one hybridoma cell clone was found to be directed against the two large subunits of the enzyme. The absence of antibodies directed against proteins possibly contaminating the antigens used for immunization allowed us to identify RNA polymerase unequivocally in interbands and puffs of polytene chromosomes. Within a single heat shock puff (87C1) RNA polymerase was found to be clustered in two separate areas suggesting two distint regions of RNA polymerase activity in this puff.Abbreviations FITC
fluorescein isothiocyanate
- PAGE
polyacrylamide gel electrophoresis
- PBS
phosphate buffered saline
- SDS
sodium dodecyl sulfate
- Enzyme
DNA-dependent RNA polymerase or nucleotide-triphosphate
- RNA
nucleotidyltransferase (EC 2.7.7.6) 相似文献
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RNA polymerase I was purified from chromatin isolated from auxin-treated soybean hypocotyl. Purification was achieved by using Agarose A-1.5m gel filtration, DEAE-cellulose, CM-sephadex, and phosphocellulose chromatography, and sucrose density gradient centrifugation. With denatured calf thymus DNA as template, the enzyme has a high specific activity (200-300 nmol/mg/30 min at 28 degrees C) which is comparable to other RNA polymerase I enzymes purified from animals and yeast. While the gel profiles indicate that purification to homogeneity (greater than 90%) may not have been achieved, the enzyme appears to be composed of possibly 7 subunits, several of which are similar to the subunits of yeast RNA polymerase I. The putative subunits and molar ratios are 183 000 (1), 136 000 (1), 50 000 (0.5), 46 000 (0.5), 40 000 (0.5), 33 000 (0.2), and 28 000 (2). The purified enzyme strongly prefers a completely denatured template such as poly(dC). 相似文献
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《Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis》1976,418(3):344-357
RNA polymerase I was purified from chromatin isolated from auxintreated soybean hypocotyl. Purification was achieved by using Agarose A-1.5m gel filtration, DEAE-cellulose, CM-sephadex, and phosphocellulose chromatography, and sucrose density gradient centrifugation. With denatured calf thymus DNA as template, the enzyme has a high specific activity (200–300 nmol/mg/30 min at 28°C) which is comparable to other RNA polymerase I enzymes purified from animals and yeast. While the gel profiles indicate that purification to homogeneity (greater than 90%) may not have been achieved, the enzyme appears to be composed of possibly 7 subunits, several of which are similar to the subunits of yeast RNA polymerase I. The putative subunits and molar ratios are 183 000 (1), 136 000 (1), 50 000 (0.5), 46 000 (0.5), 40 000 (0.5), 33 000 (0.2), and 28 000 (2). The purified enzyme strongly prefers a completely denatured template such as poly(dC). 相似文献
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Purification and characterization of the DNA-dependent RNA polymerase from Clostridium acetobutylicum.
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The DNA-dependent RNA polymerase (EC 2.7.7.6) from Clostridium acetobutylicum DSM 1731 has been purified to homogeneity and characterized. The purified enzyme was composed of four subunits and had a molecular mass of 370,000 Da. Western immunoblot analysis with polyclonal antibodies against the sigma 70 subunit of Escherichia coli RNA polymerase identified the 46,000-Da subunit as an immunologically and probably functionally related protein. The other three subunits of 128,000, 117,000, and 42,000 Da are tentatively analogous to the beta, beta', and alpha subunits, respectively, of other eubacterial RNA polymerases. The RNA polymerase activity was completely dependent on Mg2+, nucleoside triphosphates, and a DNA template. The presence of Mg2+ or Mn2+ in buffers used for purification or storage caused irreversible inactivation of the RNA polymerase. 相似文献
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Atomic absorption spectroscopy demonstrated that highly purified RNA polymerase II from the yeast Saccharomyces cerevisiae binds seven zinc ions. This number agrees with the number of potential zinc-binding sites among the 12 different subunits of the enzyme and with our observation that the ninth largest subunit alone is able to bind two zinc ions. The zinc-binding motif in the largest subunit of the enzyme was investigated using mutagenic analysis. Altering any one of the six conserved residues in the zinc-binding motif conferred either a lethal or conditional phenotype, and zinc blot analysis indicated that mutant forms of the domain had a 2-fold reduction in zinc affinity. Mutations in the zinc-binding domain reduced RNA polymerase II activity in cell-free extracts, even though protein blot analysis indicated that the mutant subunit was present in excess of wild-type levels. Purification of one mutant RNA polymerase revealed a subunit profile that was wild-type like with the exception of two subunits not required for core enzyme activity (Rpb4p and Rpb7p), which were missing. Core activity of the mutant enzyme was reduced 20-fold. We conclude that mutations in the zinc-binding domain can reduce core activity without altering the association of any of the subunits required for this activity. 相似文献
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Quantification of DNA-dependent RNA polymerase subunits and initiation factor(s) by antibody-linked polymerase assays 总被引:2,自引:0,他引:2
S Lerbs-Mache 《FEBS letters》1988,234(2):392-394
The antibody-linked polymerase assay is a method which allows one to assign RNA polymerase activity to SDS-denatured polypeptides on nitrocellulose membranes using antibodies which were raised against only partially purified polymerase preparations. Here we show that with this method not only enzyme subunits but also initiation factor(s) can be determined in crude homogenates. Moreover the determination is quantitative. Therefore changes in the amount of individual polymerase subunits and factor(s) can be visualized within different crude homogenates. 相似文献