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1.
We have identified the gene encoding the Caulobacter crescentus principal sigma subunit, RpoD. The rpoD gene codes for a polypeptide of 653 amino acids with a predicted molecular mass of 72,623 Da (sigma 73). The C. crescentus sigma subunit has extensive amino acid sequence homology with the principal sigma factors of a number of divergent procaryotes. In particular, the segments designated region 2 that are involved in core polymerase binding and promoter recognition were identical among these bacteria despite the fact that the -10 region recognized by the C. crescentus sigma 73 differs significantly from that of the other bacteria. Thus, it appears that additional sigma factor regions must be involved in -10 region recognition. This conclusion was strengthened by a heterologous complementation assay in which C. crescentus sigma 73 was capable of complementing the Escherichia coli rpoD285 temperature-sensitive mutant. Furthermore, C. crescentus sigma 73 conferred new specificity on the E. coli RNA polymerase, allowing the expression of C. crescentus promoters in E. coli. Thus, the C. crescentus sigma 73 appears to have a broader specificity than does the sigma 70 of the enteric bacteria. 相似文献
2.
Asymmetric segregation of heat-shock proteins upon cell division in Caulobacter crescentus 总被引:15,自引:0,他引:15
Three Caulobacter crescentus heat-shock proteins were shown to be immunologically related to the Escherichia coli heat-shock proteins GroEL, Lon and DnaK. A fourth heat-shock protein was detected with antibody to the C. crescentus RNA polymerase. This 37,000 Mr heat-shock protein might be related to the E. coli 32,000 Mr heat-shock sigma subunit. The synthesis of the major C. crescentus RNA polymerase sigma factor was not induced by heat shock. The E. coli GroEL protein and the related protein from C. crescentus were also induced by treatment with hydrogen peroxide. Like some of the proteins in the heat-shock protein families of Drosophila and yeast, the four heat-shock proteins in C. crescentus were found to be regulated developmentally under normal conditions. All four proteins were synthesized in the predivisional cell, but the progeny showed cell type-specific bias in the level of enhanced synthesis after heat shock. The 92,000 Mr Lon homolog and the 37,000 Mr RNA polymerase subunit were preferentially synthesized in the stalked cell, whereas the synthesis of the 62,000 Mr GroEL homolog was enhanced in the progeny swarmer cell. Furthermore, the four heat-shock proteins synthesized in the predivisional cell were partitioned in a specific manner upon cell division. The stalked cell, which initiates chromosome replication immediately upon division, received the Lon homolog, the DnaK homolog and the 37,000 Mr RNA polymerase subunit. The GroEL homolog, however, was distributed equally to both the stalked cell and the swarmer cell. These results provide access to the functions of C. crescentus heat-shock proteins under both normal and stress conditions. They also allow an investigation of the regulatory signals that modulate the asymmetric distribution of proteins and their subsequent cell type-specific expression in the initial stages of a developmental program. 相似文献
3.
RNA polymerase from Rhizobium japonicum 总被引:32,自引:0,他引:32
4.
During the purification of RNA polymerase from Xanthomonas campestris pv. oryzae, a new subunit named k was found to be associated with this enzyme. The removal of subunit k from holoenzyme by DEAE-cellulose column chromatography results in a decrease in specific activity of the enzyme. The readdition of subunit k to subunit k-depleted holoenzyme results in restoration of enzymatic activity. Subunit k increase the activity of RNA polymerase; the activation was in proportion to the concentration of subunit k added. Antiserum against holoenzyme devoid of subunit k was prepared. This antiserum did not react with purified subunit k; therefore, subunit k may not be the proteolytic fragment of the beta, beta', sigma, or alpha subunit. When this antiserum was used to precipitate RNA polymerase obtained from a crude extract of bacterial cells, subunit k was coprecipitated as determined by sodium dodecyl sulfate gel electrophoretic analysis. The molecular mass of subunit k is approximately 29 kDa, and the molar ratio of beta:beta':sigma:alpha:k was estimated to be 1:1:1:2:4. When native Xp10 DNA was used as template, subunit k stimulated subunit k-depleted holoenzyme, but not core enzyme. When the synthetic polynucleotide poly[d(A-T)] was used, subunit k activated both subunit k-depleted holoenzyme and core enzyme. Subunit k also activated the binding of RNA polymerase to template DNA. 相似文献
5.
Polyadenylic acid synthesis activity of purified DNA-dependent RNA polymerase from Caulobacter 总被引:2,自引:0,他引:2
Characterization of purified DNA-dependent RNA polymerase (EC 2.7.7.6) of Caulobacter crescentus, strain CB15 has led to the conclusion that this enzyme catalyzes poly(A) synthesis in the absence of template. Poly(A) synthetase activity co-purifies with both holoenzyme and core polymerase on DNA-cellulose columns, and core polymerase purified to 98% homogeneity by glycerol gradient centrifugation is still capable of catalyzing poly(A) polymerization. Both RNA synthesis and poly(A) polymerization activities are sensitive to rifampicin. In addition, RNA polymerase purified from partially rifampicin-sensitive mutants exhibits the same partial sensitivity in vitro to the drug in the synthesis of RNA and poly(A). The enzyme used in these studies was prepared by a simple method which allows a high yield of pure RNA polymerase from large batches of exponential cells. The procedure includes high speed centrifugation of cell extracts, DEAE-cellulose column, DNA-affinity chromatography, and low salt glycerol gradient centrifugation. Holoenzyme can be resolved into core and sigma subunit by either DNA-cellulose chromatography or glycerol gradient centrifugation, and the latter step allows recovery of pure sigma factor. 相似文献
6.
DNA-directed RNA polymerase was solubilized from total HeLa cells. Three distinct classes of the enzyme could be clearly differentiated by their sensitivity toward alpha-amanitin. While form A is completely resistant to high concentrations (133 mug/ml) of this toxin, enzyme B is highly sensitive and is completely inhibited by concentrations of 0.1 mug/ml. In contrast, RNA polymerase C shows an intermediate behaviour (50% inhibition at 30% mug/ml). Separation of the three individual enzymes was achieved by chromatography on DEAE-cellulose (to separate enzyme B from A and C) and DEAE-Sephadex (to separate polymerase A from C). All three RNA polymerases were subsequently purified by phosphocellulose chromatography followed by sedimentation through glycerol gradients. Analysis of the purified enzymes by gel electrophoresis under denaturating conditions showed that the A enzyme consists of five subunits with molecular weights of 185, 128, 65, 41 and 32 X 10(3). In contrast, polymerase B is composed of seven subunits in variable stoichiometry with molecular weights of 215, 175, 145, 123, 68, 43 and 31 X 10(3) respectively. The subunit structure of enzyme C is not entirely clear at present and remains to be established. In addition, RNA polymerase activities were solubilized from mitotic and middle-S phase cells in comparison to controls. With respect to amounts and/or activities of all three RNA polymerases A,B and C no significant differences were detectable between logarithmically growing, mitotic and middle-S phase cells. 相似文献
7.
The nucleoside triphosphate–ribonucleic acid nucleotidyltransferase (EC 2.7.7.6) of Agrobacterium tumefaciens (Smith and Townsend) Conn. Purification and properties of the enzyme from the tumorigenic strain B6806
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U. C. Knopf 《The Biochemical journal》1974,143(3):511-520
The RNA nucleotidyltransferase (RNA polymerase) of the plant-tumorigenic bacterium Agrobacterium tumefaciens was purified. The method involves the disruption of the bacterial cells with glass beads in a Waring Blendor, treatment with DEAE-cellulose, fractionation with (NH(4))(2)SO(4), protamine sulphate precipitation, DEAE-cellulose column chromatography and either glycerol-gradient centrifugation or phosphocellulose chromatography. The subunit structure of the highly purified enzyme is similar to, although not identical with, the RNA nucleotidyltransferase of Escherichia coli. It can be described as beta', beta, chi(1) and alpha (mol.wts. 160000, 150000, 98000, and 41000+/-10% respectively). chi(1) is the temporary designation for a protein subunit, which might have the same functions as the sigma subunit in E. coli. The enzyme of A. tumefaciens is rifampicin-sensitive, has a temperature optimum in vitro of 41+/-1 degrees C and a pH optimum of 8.2+/-0.1. Mg(2+) and Mn(2+) are activators. The enzyme transcribes with different efficiencies artificial, viral, bacterial, plant and animal templates. 相似文献
8.
Purification and partial characterization of the DNA-dependent RNA polymerase from Rickettsia prowazekii 总被引:3,自引:1,他引:2
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The DNA-dependent RNA polymerase was purified from Rickettsia prowazekii, an obligate intracellular bacterial parasite. Because of limitation of available rickettsiae, the classical methods for isolation of the enzyme from other procaryotes were modified to purify RNA polymerase from small quantities of cells (25 mg of protein). The subunit composition of the rickettsial RNA polymerase was typical of a eubacterial RNA polymerase. R. prowazekii had beta' (148,000 daltons), beta (142,000 daltons), sigma (85,000 daltons), and alpha (34,500 daltons) subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The appropriate subunits of the rickettsial RNA polymerase bound to polyclonal antisera against Escherichia coli core polymerase and E. coli sigma 70 subunit in Western blots (immunoblots). The enzyme activity was dependent on all four ribonucleoside triphosphates, Mg2+, and a DNA template. Optimal activity occurred in the presence of 10 mM MgCl2 and 50 mM NaCl. Interestingly, in striking contrast to E. coli, approximately 74% of the rickettsial RNA polymerase activity was associated with the rickettsial cell membrane at a low salt concentration (50 mM NaCl) and dissociated from the membrane at a high salt concentration (600 mM NaCl). 相似文献
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Plastid genes are transcribed by DNA-dependent RNA polymerase(s), which have been incompletely characterized and have been examined in a limited number of species. Plastid genomes contain rpoA, rpoB, rpoC1, and rpoC2 coding for alpha, beta, beta', and beta" RNA polymerase subunits that are homologous to the alpha, beta, and beta' subunits that constitute the core moiety of RNA polymerase in bacteria. However, genes with homology to sigma subunits in bacteria have not been found in plastid genomes. An antibody directed against the principal sigma subunit of RNA polymerase from the cyanobacterium Anabaena sp. PCC 7120 was used to probe western blots of purified chloroplast RNA polymerase from maize, rice, Chlamydomonas reinhardtii, and Cyanidium caldarium. Chloroplast RNA polymerase from maize and rice contained an immunoreactive 64-kD protein. Chloroplast RNA polymerase from C. reinhardtii contained immunoreactive 100- and 82-kD proteins, and chloroplast RNA polymerase from C. caldarium contained an immunoreactive 32-kD protein. The elution profile of enzyme activity of both algal chloroplast RNA polymerases coeluted from DEAE with the respective immunoreactive proteins, indicating that they are components of the enzyme. These results provide immunological evidence for sigma-like factors in chloroplast RNA polymerase in higher plants and algae. 相似文献
12.
The quaternary structures of Escherichia coli DNA-dependent RNA polymerase holenzyme (alpha 2 beta beta' sigma) and core enzyme (alpha 2 beta beta') have been investigated by chemical cross-linking with a cleavable bifunctional reagent, methyl 4-mercaptobutyrimidate, and noncleavable reagents, dimethyl suberimidate and N,N'-(1,4-phenylene)bismaleimide. A model of the subunit organization deduced from cross-linked subunit neighbors identified by dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the large beta and beta' subunits constitute the backbone of both core and holoenzyme, while sigma and two alpha subunits interact with this structure along the contact domain of beta and beta' subunits. In holoenzyme, sigma subunit is in the vicinity of at least one alpha subunit. The two alpha subunits are close to each other in holoenzyme, core enzyme, and the isolated alpha 2 beta complex. Cross-linking of the "premature" core and holoenzyme intermediates in the in vitro reconstitution of active enzyme from isolated subunits suggests that these species are composed of subunit complexes of molecular weight lower than that of native core and holoenzyme, respectively. The structural information obtained for RNA polymerase and its subcomplexes has important implications for the enzyme-promoter recognition as well as the mechanism of subunit assembly of the enzyme. 相似文献
13.
Sequence-specific interaction of sigma subunit of E. coli RNA polymerase with DNA. 总被引:2,自引:1,他引:1
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W Stender 《Nucleic acids research》1980,8(15):3459-3466
The interaction of sigma subunit of E. coli RNA polymerase with DNA, either double or single-stranded, and with two inhibitors of RNA synthesis was investigated by using antibodies directed against the subunit. Free sigma subunit was shown to interact with poly(dA), poly(dT), poly(dAC).poly(dGT), T7 DNA and, to a lesser degree, with lambda DNA. When the sigma subunit forms part of the holo enzyme, sigma also interacts with poly(dG).poly(dC). Rifampicin and streptolydigin interact with sigma in the holo enzyme and with free and core bound sigma subunit, respectively. The results suggest that sigma recognizes mainly AC-GT-sequences in double-stranded DNA. The findings are correlated with the base composition in RNA polymerase binding regions of promoters and suggest at least a general interaction between sigma subunit and single-stranded DNA in open complexes. 相似文献
14.
Highly purified Escherichia coli RNA polymerase contains a small subunit termed omega that has a molecular mass of 10 105 Da and is comprised of 91 amino acids. E. coli strains lacking omega (omega-less) are viable, but exhibit a slow-growth phenotype. Renaturation of RNA polymerase isolated from an omega-less mutant, in the presence of omega, resulted in maximum recovery of activity. The omega-less RNA polymerase from omega-less strains recruits the chaperonin, GroEL (unlike the wild-type enzyme), suggesting a structural deformity of the mutant enzyme. The GroEL-containing core RNA polymerase interacts efficiently with sigma70 to generate the fully functional holoenzyme. However, when GroEL was removed, the enzyme was irreversibly nonfunctional and was unable to bind to sigma70. The damaged enzyme regained activity after going through a cycle of denaturation and reconstitution in the presence of omega or GroEL. GroES was found to have an inhibitory effect on the core-sigma70 association unlike the omega subunit. The omega subunit may therefore be needed for stabilization of the structure of RNA polymerase. 相似文献
15.
RNA polymerase II subunit RPB4 is essential for high- and low-temperature yeast cell growth. 总被引:44,自引:19,他引:25
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RPB4 encodes the fourth-largest RNA polymerase II subunit in Saccharomyces cerevisiae. The RPB4 gene was cloned and sequenced, and its identity was confirmed by amino acid sequence analysis of tryptic peptides from the purified subunit. The RPB4 DNA sequence predicted a protein of 221 amino acids with a molecular mass of 25,414 daltons. The central 100 amino acids of the RPB4 protein were found to be similar to a segment of the major sigma subunit in Escherichia coli RNA polymerase. Deletion of RPB4 produced cells that were heat and cold sensitive but could grow, albeit slowly, at intermediate temperatures. RNA polymerase II lacking the RPB4 subunit exhibited markedly reduced activity in crude extracts in vitro. The RPB4 subunit, although not essential for mRNA synthesis or enzyme assembly, was essential for normal levels of RNA polymerase II activity and indispensable for cell viability over a wide temperature range. 相似文献
16.
Footprint of the sigma protein 总被引:2,自引:0,他引:2
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19.
Purification and immunological analysis of RNA polymerase II from Caenorhabditis elegans 总被引:1,自引:0,他引:1
We describe a rapid procedure for obtaining highly purified RNA polymerase II from the nematode Caenorhabditis elegans. The structure of the enzyme was examined by denaturing gel electrophoresis and found to consist of three large polypeptides (molecular weights 200,000, 175,000, and 135,000) and eight smaller polypeptides (molecular weights 29,500, 20,000, 16,000, 15,000, 13,000, 11,500, 10,500, and 9,500). As observed for the analogous enzyme from other organisms, the 175,000 polypeptide (II175) appeared to be a degraded form of the 200,000 polypeptide (II200). The structure of nematode RNA polymerase II closely resembles that of the corresponding enzyme from other animals. Four of its larger subunits shared antigenicity with Drosophila RNA polymerase II. Antibody raised against purified RNA polymerase II reacted with several enzyme subunits in "Western" blots of purified polymerase and impure enzyme fractions. Immunofluorescence staining was used to visualize RNA polymerase II in the nuclei of a nematode squash preparation and the nucleoplasm of cultured mammalian cells. 相似文献
20.
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