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Monoclonal antibodies as probes of the topological arrangement of the alpha subunits of Escherichia coli RNA polymerase 总被引:4,自引:0,他引:4
Three monoclonal anti-alpha antibodies were used to study the properties of the alpha subunit of Escherichia coli RNA polymerase. None of the monoclonal antibodies inhibited the d(A-T)n-directed synthesis of r(A-U)n. Reassembly of the RNA polymerase core was blocked by mAb 129C4 or mAb 126C6 while no effect was observed with mAb 124D1. The conversion of premature to mature core was partially inhibited by mAb 129C4 and almost totally inhibited by mAb 126C6. The data suggest that during the course of core assembly at least one of the alpha subunits undergoes conformational changes. The increase in affinity of mAb 126C6 for assembled alpha compared with free alpha also implies that alpha undergoes conformational changes during RNA polymerase assembly. Double antibody binding studies showed that the epitopes for mAb 124D1 and mAb 129C4 are available on only one of the alpha subunits in RNA polymerase. It would appear that the relevant domain on one of the alpha subunits in RNA polymerase is well exposed whereas this domain on the second alpha subunit is shielded by interaction with regions of the large beta and beta' subunits. The alpha domain in which the epitope for mAb 126C6 resides is not impeded by subunit interactions in the RNA polymerase. The data obtained also suggest that in the holoenzyme the sigma subunit may be positioned close to one of the alpha subunits, probably to the more exposed alpha. The alpha beta complex is the minimal stable subassembly since one of the alpha subunits dissociates from the alpha 2 beta complex following binding of any of the monoclonal antibodies studied.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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Antibodies against the subunits of E. coli RNA polymerase as probes for subunit-specific binding of DNA and other ligands. 总被引:2,自引:2,他引:0
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W Stender 《Nucleic acids research》1980,8(6):1405-1420
Antibodies against the isolated subunits alpha, beta, and beta' of DNA-dependent RNA polymerase from E. coli have been prepared. They have been used to compare the extent of antibody-binding, as measured by complement fixation, to the isolated subunits and to the intact enzyme, in the absence and presence of ligands, such as inhibitors, nucleotides, nucleosides, oligo- and poly-nucleotides, and DNA of different composition. In many cases the results show a subunit-specific dependence of complement fixation upon the presence of a ligand and suggest a functional topography of the interaction between the subunits alpha, beta, and beta' of RNA polymerase and defined nucleotide sequences and small ligands. 相似文献
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TSH is a heterodimeric glycoprotein hormone, whose dissociated subunits are without biological activity. This has precluded the assessment of the relative contribution of each subunit to hormone action. We have raised anti-idiotypes to monoclonal antibodies specific, respectively, for the alpha and beta hTSH subunits. The anti-beta anti-idiotype inhibited 125I-hTSH binding to the beta subunit-specific monoclonal quantitatively, whereas 125I-hTSH binding to the alpha subunit-specific monoclonal was not inhibited by anti-alpha anti-idiotypes, suggesting that only the former is an "internal image" anti-idiotype. Neither of the two anti-idiotypes nor equimolar mixtures thereof inhibited 125I-bTSH binding to thyroid membranes, even though radiolabelled anti-idiotypes showed saturable binding to thyroid plasma membrane which was inhibited 41-65% by bTSH. Each anti-idiotype alone caused 9% inhibition (compared to 50% by NRIgG) of thyroid plasma membrane adenylate cyclase. Equimolar mixtures (125 micrograms/ml IgG of each anti-idiotype) induced enzyme activity equivalent to 85% of that of 250 mU/ml of TSH. The TSH-like action of the two anti-idiotypes was also reflected in their capacity to increase (450% by 250 micrograms/ml IgG compared to normal rabbit IgG) the uptake of 131I into isolated thyrocytes and to promote the organization of such cells into follicular structures. At 250 micrograms/ml, anti-beta anti-idiotype promoted the organization of small follicles and only at a concentration of 500 micrograms/ml did it enhance 131I uptake. 相似文献
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V D Axelrod R M Vartikyan V A Aivazashvili R S Beabealashvili 《Nucleic acids research》1978,5(10):3549-3563
Termination of RNA synthesis with 3'-O-Methylnucleoside 5'-triphosphates have been studied using E. coli RNA polymerase holoenzyme and poly [d(A-T)] as well as unfractionated T7 D delta III DNA as templates. It was shown that the termination can be used for DNA sequencing. A sequence of a part of RNA synthesized from AI promoter of the DNA have been determined. Syntheses of four 3'-O-Methylnucleoside 5'-triphosphates are described. 相似文献
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Ebright RH 《Journal of molecular biology》2000,304(5):687-698
Bacterial RNA polymerase and eukaryotic RNA polymerase II exhibit striking structural similarities, including similarities in overall structure, relative positions of subunits, relative positions of functional determinants, and structures and folding topologies of subunits. These structural similarities are paralleled by similarities in mechanisms of interaction with DNA. 相似文献
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RNA polymerase III (C) and its transcription factors. 总被引:25,自引:0,他引:25
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The distribution of DNA-dependent RNA polymerase in Escherichia coli was analysed by measuring enzyme subunits in nucleoid (folded chromosome) and cytoplasm. Two independent methods, two-dimensional polyacrylamide gel electrophoresis of total proteins and sodium dodecyl sulphate/polyacrylamide gel electrophoresis of antibody precipitates, gave essentially the same results; with wild-type cells growing at a doubling time of 70 minutes, about two-thirds of the core enzyme but little σ subunit are present in the nucleoid. Pulse-chase experiments indicated that the distribution of the pulse-labelled proteins was at equilibrium within 1·5 minutes for β′, 5 minutes for β, and 15 minutes for α subunit. This order of appearance of the newly synthesized core subunits into the nueleoid is in good agreement with that into complete enzyme structure. This finding, together with the known sequence of subunit assembly (2α → α2 → α2β → α2ββ′ → E), indicates that the assembly of RNA polymerase takes place in the cytoplasm. In concert with the conclusion, the amounts of pulse-labelled subunits in the cytoplasm of temperature-sensitive assembly defective mutants coincide well with those of intermediate subassemblies accumulated in the mutant cells. However, it is not known if the premature core is activated in cytoplasm prior to binding to the nucleoid or shortly after association with the nucleoid. 相似文献
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Isolation and characterization of monoclonal antibodies directed against two subunits of rabbit poxvirus-associated, DNA-directed RNA polymerase. 总被引:13,自引:9,他引:4
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A library of monoclonal antibodies directed against individual proteins of the rabbit poxvirus (RPV) virion within a complex immunogenic mixture has been generated through the use of in vivo and in vitro immunization regimens. The relative efficacies of the two procedures were compared. Based on immunoblot analysis, the in vitro immunization regimen led both to a wider variety of monoclonal antibodies to different proteins and to a larger number of antibodies directed against proteins of higher molecular weights. Each method, however, has advantages, and the two procedures appear to be complementary. A simple method to recognize antibodies directed against the virion DNA-directed RNA polymerase was developed. Monoclonal antibodies directed against two subunits (137 and 34 kilodaltons [kDa]) of the RNA polymerase were identified and used to study the biogenesis of the enzyme and to map the two corresponding genes within the viral genome by using an RPV DNA library cloned into the lambda gtll expression vector. Both proteins are synthesized late in the infectious cycle and are restricted totally to the cytoplasm. Preliminary mapping data place the genes encoding the 137-kDa protein within the HindIII H fragment, whereas the gene for the 34-kDa protein is located within the left most region of the HindIII A fragment. 相似文献
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Monoclonal antibodies to double-stranded RNA as probes of RNA structure in crude nucleic acid extracts. 总被引:9,自引:1,他引:8
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J Schnborn J Oberstrass E Breyel J Tittgen J Schumacher N Lukacs 《Nucleic acids research》1991,19(11):2993-3000
We describe four monoclonal antibodies (MAB) which specifically recognize double-stranded RNA (dsRNA) together with their use in new methods for detecting and characterizing dsRNA in unfractionated nucleic acid extracts. The specificity of the antibodies was analyzed using a panel of 27 different synthetic and naturally occurring nucleic acids. All four antibodies reacted in a highly specific manner with long dsRNA helices, irrespective of their sequence; no binding to single-stranded RNA homopolymers or to DNA or RNA-DNA hybrids was observed. The apparent affinity of the antibodies to short (less than or equal to 11 bp) RNA helices was very low in all test systems used: only background levels of binding were obtained on single-stranded RNA species which contain double-helical secondary structures (e.g. rRNA, tRNA, viroid RNA). A sandwich ELISA and a dsRNA-immunoblotting procedure have been established which allow detection and characterization of dsRNA by MAB even in the presence of a large excess of other nucleic acids. In combination with temperature-gradient gelelectrophoresis (TGGE) not only the molecular weights but also the highly characteristic Tm-values of conformational transitions of individual dsRNA species could be determined by immunoblotting. An example of the general use of these methods for the detection of plant virus infections is demonstrated with groundnut rosette virus (GRV) dsRNAs. We were able to estimate the dsRNA content of infected leaves, identify the dsRNA species present in crude extracts and to determine the Tm- values of GRV dsRNA-3. 相似文献
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M Bustin 《Nucleic acids research》1978,5(3):925-932
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Monoclonal antibodies to bovine UDP-galactosyltransferase. Characterization, cross-reactivity, and utilization as structural probes 总被引:2,自引:0,他引:2
J T Ulrich J R Schenck H G Rittenhouse N L Shaper J H Shaper 《The Journal of biological chemistry》1986,261(17):7975-7981
A series of mouse monoclonal antibodies has been developed against a soluble form of bovine UDP-galactose:N-acetylglucosamine galactosyltransferase purified to apparent chemical homogeneity by a combination of affinity and immunoadsorption chromatography. The purified enzyme consists of two molecular mass variants of 42 and 48 kDa. Individual monoclonal antibodies were selected for by their ability to recognize immobilized affinity-purified galactosyltransferase and were not reactive against bovine alpha-lactalbumin and bovine immunoglobulins. Based on competitive binding assays and Western blot analysis with either galactosyltransferase or lactose synthetase (covalently cross-linked alpha-lactalbumin galactosyltransferase), these monoclonal antibodies can be subdivided into four groups. Group A (3 clones) recognize an epitope at or near the alpha-lactalbumin binding site. In addition, this group is cross-reactive with soluble galactosyltransferase from human milk and pleural effusion. Group B (6 clones) and D (1 clone) appear to recognize two different epitopes on the 6-kDa fragment which is released when the 48-kDa galactosyltransferase polypeptide is converted to the 42-kDa form, apparently by proteolysis. Groups A and C (1 clone) recognize epitopes found on both the 48- and 42-kDa polypeptide. Interestingly, immunofluorescence studies indicate that only two monoclonal antibody groups (C and D) are able to decorate membrane-bound galactosyltransferase (Golgi-associated) in formalin-fixed, methanol-, or detergent-permeabilized cells. Thus, these groups of monoclonal antibodies appear to identify four separate structural/functional domains on soluble galactosyltransferase, two of which are not readily accessible for binding in situ. 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