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1.
OmpT is a protease present in the outer membrane of Escherichia coli. The enzyme was overexpressed without its signal sequence in E. coli using a T7 system, resulting in the accumulation of OmpT as inclusion bodies. After solubilization of the inclusion bodies in urea, the protein could be folded in vitro by dilution in the presence of detergent n-dodecyl-N, N-dimethyl-1-ammonio-3-propanesulphonate. The addition of lipopolysaccharide to the protein was essential to obtain active enzyme. The correctly folded protein was purified to homogeneity by ion exchange chromatography with a 57% overall yield. Autoproteolysis between Lys217-Arg218 was a major problem during purification, but degradation could be abolished by introducing the mutations G216K and K217G. A novel fluorimetric assay using the internally quenched substrate Abz-Ala-Arg-Arg-Ala-Tyr(NO2)-NH2 (where Abz is o-aminobenzoyl and Tyr(NO2) is 3-nitrotyrosine) enabled the determination of the kinetic parameters. The wild-type enzyme has an affinity Km of 0.4 microM for the substrate and a turnover number kcat of 40 s-1. The Km and kcat for the double variant were 1.1 microM and 1.6 s-1, respectively. The pH profiles of the wild type and variant were identical, showing optimal activity at pH 6.5 and pKa values of 5.6 and 7.5, respectively. Circular dichroism spectra of both enzymes indicated a high content of beta-strand conformation, and on that basis a beta-barrel topology model is proposed. 相似文献
2.
Induction of the adaptive response by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) caused a decrease in the UV-mediated expression of both recA and sfiA genes but not of the umuDC gene. On the other hand, the adaptive response did not affect the temperature-promoted induction of SOS response in a RecA441 mutant. The inhibitory effect on the UV-triggered expression of the recA and sfiA genes was not dependent on either the alkA gene or the basal level of RecA protein, but rather required the ada gene. Furthermore, an increase in the level of the Ada protein, caused by the runaway plasmid pYN3059 in which the ada gene is regulated by the lac promoter, inhibited UV-mediated recA gene expression even in cells to which the MNNG-adaptive treatment had not been applied. This inhibitory effect of the adaptive pretreatment was not observed either in RecBC- strains or in RecBC mutants lacking exonuclease V-related nuclease activity. However, RecF- mutants showed an adaptive response-mediated decrease in UV-promoted induction of the recA gene. 相似文献
3.
Escherichia coli protein X is the recA gene product. 总被引:16,自引:0,他引:16
Escherichia coli protein X is known to be made in large amounts following DNA damage or inhibition of DNA replication. We have shown that it is identical to the recA gene product by partial proteolytic digestion of the radiochemically pure proteins and analysis by electrophoresis on polyacrylamide-sodium dodecyl sulfate gels. 相似文献
4.
Summary Derivatives of the Escherichia coli drug resistance plasmid pMB-9 were constructed which contain the promotor from the lactose operon of E. coli fused to the araC gene of E. coli. E. coli possessing these plasmids contain about 50 times as much of the araC gene product as do cells with a wild-type araC gene and promotor. 相似文献
5.
The lpd gene product functions as the L protein in the Escherichia coli glycine cleavage enzyme system. 总被引:5,自引:5,他引:0
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The lpd-encoded lipoamide dehydrogenase, common to the pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes, also functions as the lipoamide dehydrogenase (L protein) in the Escherichia coli glycine cleavage (GCV) enzyme complex. Inducible GCV enzyme activity was not detected in an lpd deletion mutant; lpd+ transductants had normal levels of inducible GCV enzyme activity. A serA lpd double mutant was unable to utilize glycine as a serine source and lacked detectable GCV enzyme activity, the phenotype of a serA gcv mutant. Transformation of the double mutant with a plasmid encoding a functional lpd gene restored the ability of the mutant to use glycine as a serine source and restored inducible GCV enzyme activity to normal levels. The presence of acetate and succinate in the growth medium of a strain wild type for lpd and gcv resulted in a 50% reduction in inducible GCV enzyme activity. Enzyme levels were restored to normal under these growth conditions when the strain was transformed with a plasmid encoding a functional lpd gene. 相似文献
6.
The Escherichia coli Ada protein repairs O6-methylguanine residues and methyl phosphotriesters in DNA by direct transfer of the methyl group to a cysteine residue located in its C- or N-terminal domain, respectively. Methyl transfer to the N-terminal domain causes it to acquire a sequence-specific DNA binding activity, which directs binding to the regulatory region of several methylation-resistance genes. In this paper we show that the N-terminal domain of Ada contains a high-affinity binding site for a single zinc atom, whereas the C-terminal domain is free of zinc. The metal-binding domain is apparently located within the first 92 amino acids of Ada, which contains four conserved cysteine residues. We propose that these four cysteines serve as the zinc ligand residues, coordinating the metal in a tetrahedral arrangement. One of the putative ligand residues, namely, Cys69, also serves as the acceptor site for a phosphotriester-derived methyl group. This raises the possibility that methylation-dependent ligand reorganization about the metal plays a role in the conformational switching mechanism that converts Ada from a non-sequence-specific to a sequence-specific DNA-binding protein. 相似文献
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9.
In vitro catalysis of oxidative folding of disulfide-bonded proteins by the Escherichia coli dsbA (ppfA) gene product. 总被引:8,自引:0,他引:8
It was shown previously that the Escherichia coli gene ppfA (dsbA) encodes a periplasmic protein, and its inactivation leads to a deficiency in disulfide bond formation of envelope proteins (Kamitani, S., Akiyama, Y., and Ito, K. (1992) EMBO J. 11, 57-62; Bardwell, J. C. A., McGovern, K., and Beckwith, J. (1991) Cell 67, 581-589). The DsbA/PpfA protein was overproduced, purified, and examined for its activities in vitro. Its abundance in a wild-type cell was estimated to be about 850 molecules which probably exist as homodimers as suggested by size exclusion chromatography. Purified DsbA markedly stimulated disulfide bond formation of E. coli alkaline phosphatase, either in vitro synthesized or purified and denatured, as well as of reduced bovine ribonuclease A. The DsbA-catalyzed rapid disulfide bond formation occurred after a lag period which appeared to be determined by the redox state of the reaction mixture and concentration of DsbA. Inclusion of higher concentrations of oxidized glutathione or DsbA shortened the lag period. We propose that DsbA, which proved to directly catalyze disulfide bond formation, may also have a role in maintaining the bacterial periplasm oxidative. 相似文献
10.
The purine regulon repressor protein, PurR, was shown to be a purine component involved in glyA regulation in Escherichia coli. Expression of glyA, encoding serine hydroxymethyltransferase activity, was elevated in a purR mutant compared with a wild-type strain. When the purR mutant was transformed with a plasmid carrying the purR gene, the serine hydroxymethyltransferase levels returned to the wild-type level. The PurR protein bound specifically to a DNA fragment carrying the glyA control region, as determined by gel retardation. In a DNase I protection assay, a 24-base-pair region was protected from DNase I digestion by PurR. The glyA operator sequence for PurR binding is similar to that reported for several pur regulon genes. 相似文献
11.
An in vitro complementation assay for the Escherichia coli uvrD gene product. 总被引:5,自引:2,他引:3
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An in vitro assay specific for the product of the uvrD gene of Escherichia coli has been developed. This assay, derived from properties of uvrD mutants revealed by in vivo experiments, is based on the necessity for a functional UvrD protein for complete rejoining of covalently closed circular DNA during the excision repair of UV-induced damage. Extracts prepared from gently lysed uvrD101 mutant cells are capable of restoring UV-damaged DNA to its covalently closed circular form when provided with a functional UvrD protein from other repair-deficient cell extracts or from partially purified protein fractions. This assay was employed to monitor the activity of the UvrD protein after several steps of fractionation. The partially purified UvrD protein does not complement extracts deficient in DNA polymerase I or temperature-sensitive in DNA ligase; it does, however, complement extracts from strains mutant at the uvrE and recL loci, which are considered allelic with the uvrD locus. 相似文献
12.
Specific endonucleolytic cleavage of the mRNA for ribosomal protein S20 of Escherichia coli requires the product of the ams gene in vivo and in vitro. 总被引:9,自引:7,他引:2
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G A Mackie 《Journal of bacteriology》1991,173(8):2488-2497
Endonucleolytic cleavage is believed to initiate the degradation of most bacterial mRNAs, but with several exceptions, the enzymes responsible have yet to be identified. Crude (S-30) or partially fractionated extracts of Escherichia coli strains with reduced exonuclease activities catalyze the cleavage of a 372-residue RNA substrate containing the sequences coding for ribosomal protein S20 to yield a number of discrete products. The major product of 147 residues is obtained in 60 to 70% yield, is coterminal with the 3' end of the substrate, and is identical to an mRNA fragment previously characterized in vivo (G. A. Mackie, J. Bacteriol. 171:4112-4120, 1989). A number of other products of 150 to 340 residues are also formed, and the cleavage sites, typically N decreases AU sequences, have been identified in the S20 mRNA substrate by Northern (RNA) blotting and primer extension. All cleavages required a native rather than a denatured RNA substrate. The rate of cutting of the S20 mRNA substrate at the site yielding the prominent 147-residue product appears to be independent of cleavages at other sites. In addition, the activity of the putative endonuclease(s) depends strongly, both in vivo and in vitro, on the product of the ams gene, which is known to influence mRNA lifetimes in vivo. Taken together, the data show that the fractionated extract described here reproduces steps in the degradation of some mRNAs which occur in living cells. 相似文献
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K Severinov A Mustaev M Kashlev S Borukhov V Nikiforov A Goldfarb 《The Journal of biological chemistry》1992,267(18):12813-12819
The 1342 amino acid long beta subunit of Escherichia coli RNA polymerase includes a dispensable region (residues 940-1040) that is absent in homologous RNA polymerase subunits from chloroplasts, eukaryotes, and archaebacteria (Borukhov, S., Severinov, K., Kashlev, M., Lebedev, A., Bass, I., Rowland, G. C., Lim, P.-P., Glass, R. E., Nikiforov, V., and Goldfarb, A. (1991) J. Biol. Chem. 266, 23921-23926). Genetic disruption of this region by in-frame deletion or insertion sensitizes the beta subunit in assembled RNA polymerase molecules to attack by trypsin. We demonstrate that RNA polymerase with the beta polypeptide cleaved in the dispensable region retains normal in vitro activity. Moreover, the RNA polymerase activity is completely restored after denaturation and reconstitution of the enzyme carrying cleaved beta subunit indicating that its carboxyl- and amino-terminal parts fold and assemble into RNA polymerase as separate entities. 相似文献
15.
Escherichia coli TonB protein is exported from the cytoplasm without proteolytic cleavage of its amino terminus 总被引:33,自引:0,他引:33
The requirement for TonB protein in a variety of membrane-related processes suggests that TonB is an envelope protein. Consistent with this suggestion, the deduced TonB amino acid sequence (Postle, K., and Good, R. F., (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 5235-5239) contains an amino-terminal region similar to leader (signal) sequences of exported proteins, although its charged region falls outside the rules which characterize these sequences (von Heijne, G. (1985) J. Mol. Biol. 184, 99-105). The deduced TonB amino acid sequence contains three potential methionine start codons in the first six codons of the open reading frame. In this report, we show, by Edman degradation of [35S]methionine-labeled protein, that TonB protein synthesized in vitro initiates at the third of these methionine codons. A method for detecting TonB synthesized in vivo has been developed that involves expression of TonB from the lambda PL promoter and pulse labeling with [35S]methionine. TonB synthesized in vivo has a chemical half-life of 10 min at 42 degrees C. It is exported from the cytoplasm, as determined by proteinase K accessibility experiments. It fractionates with spheroplasts under conditions where maltose-binding protein fractionates with the periplasm. It has the same mobility in three different polyacrylamide gel systems as TonB synthesized in vitro. We concluded that the amino terminus of TonB is uncleaved following its export from the cytoplasm and that TonB is a membrane-associated protein. Characterization of a tonB-phoA gene fusion suggests that the amino-terminal 41 amino acids of TonB are sufficient to promote export of the fusion protein and presumably TonB as well. Models for TonB orientation within the cell envelope are presented. 相似文献
16.
lon gene product of Escherichia coli is a heat-shock protein 总被引:30,自引:15,他引:15
The product of the pleiotropic gene lon is a protein with protease activity and has been tentatively identified as protein H94.0 on the reference two-dimensional gel of Escherichia coli proteins. Purified Lon protease migrated with the prominent cellular protein H94.0 in E. coli K-12 strains. Peptide map patterns of Lon protease and H94.0 were identical. A mutant form of the protease had altered mobility during gel electrophoresis. An E. coli B/r strain that is known to be defective in Lon function contained no detectable H94.0 protein under normal growth conditions. Upon a shift to 42 degrees C, however, the Lon protease was induced to high levels in K-12 strains and a small amount of protein became detectable at the H94.0 location in strain B/r. Heat induction of Lon protease was dependent on the normal allele of the regulatory gene, htpR, establishing lon as a member of the high-temperature-production regulon of E. coli. 相似文献
17.
The product of the lexC gene of Escherichia coli is single-stranded DNA-binding protein. 总被引:6,自引:2,他引:4
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Extracts from lexC113 cells could not support phage G4 DNA-dependent replication unless supplemented with single-stranded DNA-binding protein. Purified lexC113 binding protein supported synthesis in a reconstituted replication assay, using purified proteins at 30 but not at 42 degrees C, indicating that the product of the lexC113 gene is an altered single-stranded DNA-binding protein. 相似文献
18.
Identification of the Escherichia coli recN gene product as a major SOS protein. 总被引:8,自引:5,他引:3
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The recA+ lexA+-dependent induction of four Escherichia coli SOS proteins was readily observed by two-dimensional gel analysis. In addition to the 38-kilodalton (kDa) RecA protein, which was induced in the greatest amounts and was readily identified, three other proteins of 115, 62, and 12 kDa were seen. The 115-kDa protein is the product of the uvrA gene, which is required for nucleotide excision repair and has previously been shown to be induced in the SOS response. The 62-kDa protein, which was induced to high intracellular levels, is the product of recN, a gene required for recBC-independent recombination. The recA and recN genes were partially derepressed in a recBC sbcB genetic background, a phenomenon which might account for the recombination proficiency of such strains. The 12-kDa protein has yet to be identified. 相似文献
19.
Overexpression of plasmid-coded PBP 3 was analyzed in strains harboring ftsA, ftsH, pbpB (ftsI), ftsQ, ftsZ, or recA441 (Tif) mutations. Higher cellular levels of PBP 3, the pbpB gene product, could not restore septum formation of ftsA, ftsQ, ftsZ, and recA (Tif) mutants at 42 degrees C. However, filamentation in strains harboring pbpB and ftsH mutations was fully suppressed by PBP 3 overexpression. Additional observations indicated that the Y16 (ftsH) strain, not transformed with the PBP 3-overproducing plasmid, had no detectable PBP 3 in envelopes after incubation at the restrictive temperature. These results suggest that suppression of filamentation of fts strains overexpressing wild-type cell division proteins after the shift to the restrictive temperature can be a useful strategy to demonstrate in vivo interactions of cell division gene products. 相似文献
20.
DNA cleavage and degradation by the SbcCD protein complex from Escherichia coli. 总被引:5,自引:0,他引:5
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The SbcCD protein is a member of a group of nucleases found in bacteriophage T4 and T5, eubacteria, archaebacteria, yeast, Drosophila, mouse and man. Evidence from electron microscopy has revealed a distinctive structure consisting of two globular domains linked by a long region of coiled coil, similar to that predicted for the members of the SMC family. That a nuclease should have such an unusual structure suggests that its mode of action may be complex. Here we show that the protein degrades duplex DNA in a 3'-->5' direction. This degradation releases products half the length of the original duplex suggesting simultaneous degradation from two duplex ends. This may provide a link to the unusual structure of the protein since our data are consistent with recognition and cleavage of DNA ends followed by 3'-->5' nicking by two nucleolytic centres within a single nuclease molecule that releases a half length limit product. We also show that cleavage is not simply at the point of a single-strand/double-stand transition and that despite the dominant 3'-->5' polarity of degradation, a 5' single-strand can be cleaved when attached to duplex DNA. The implications of this mechanism for the processing of hairpins formed during DNA replication are discussed. 相似文献