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At least 523 records · Page 29Linked to original sources
PubMed1999

Wrapping of flanking non-operator DNA in lac repressor-operator complexes: implications for DNA looping.

In our studies of lac repressor tetramer (T)-lac operator (O) interactions, we observed that the presence of extended regions of non-operator DNA flanking a single lac operator sequence embedded in plasmid DNA produced large and unusual cooperative and anticooperative effects on binding constants (Kobs) and their salt concentration dependences for the formation of 1:1 (TO) and especially 1:2 (TO2) complexes. To explore the origin of this striking behavior we report and analyze binding data on 1:1 (TO) and 1:2 (TO2) complexes between repressor and a single O(sym) operator embedded in 40 bp, 101 bp, and 2514 bp DNA, over very wide ranges of [salt]. We find large interrelated effects of flanking DNA length and [salt] on binding constants (K(TO)obs, K(TO2)obs) and on their [salt]-derivatives, and quantify these effects in terms of the free energy contributions of two wrapping modes, designated local and global. Both local and global wrapping of flanking DNA occur to an increasing extent as [salt] decreases. Global wrapping of plasmid-length DNA is extraordinarily dependent on [salt]. We propose that global wrapping is driven at low salt concentration by the polyelectrolyte effect, and involves a very large number (>/similar 20) of coulombic interactions between DNA phosphates and positively charged groups on lac repressor. Coulombic interactions in the global wrap must involve both the core and the second DNA-binding domain of lac repressor, and result in a complex which is looped by DNA wrapping. The non-coulombic contribution to the free energy of global wrapping is highly unfavorable ( approximately +30-50 kcal mol(-1)), which presumably results from a significant extent of DNA distortion and/or entropic constraints. We propose a structural model for global wrapping, and consider its implications for looping of intervening non-operator DNA in forming a complex between a tetrameric repressor (LacI) and one multi-operator DNA molecule in vivo and in vitro. The existence of DNA wrapping in LacI-DNA interactions motivates the proposal that most if not all DNA binding proteins may have evolved the capability to wrap and thereby organize flanking regions of DNA.

Bacterial Proteins↗
PubMed2000

Strengthening the dimerisation interface of Lac repressor increases its thermostability by 40 deg. C.

We increased drastically the heat stability of Lac repressor (LacR) of Escherichia coli. Wild-type tetrameric LacR denatures irreversibly at 53 degrees C. Improving hydrophobic packing at the dimerisation interface by a single substitution increases LacR heat-resistance by 40 deg. C without abolishing inducer binding at high and low temperatures. Tetrameric LacR mutants carrying substitutions of the positively charged amino acid Lys84 by each of the hydrophobic amino acids Leu, Ile and Met resist heating to temperatures up to 93 degrees C. We performed IPTG binding assays at 80 degrees C and found the mutant Lac repressors active and, thus, the core intact. Furthermore, the activity of LacR following heating is shown at room temperature by a gel retardation assay, which demonstrates normal oligomerisation state and function of the headpiece. The same mutations (K84L/I/M) in the dimer LacR331stop, carrying a stop codon in amino acid 331, increase thermostability of the dimer from 47 degrees C to 87 degrees C. LacRK84M represses beta-galactosidase activity in vivo as well as the wild-type and is sufficiently induced to allow growth on lactose. The results with both tetramer and dimer variants of LacR indicate mutual stabilisation of the tetramerisation region and the stable core.

Allosteric Regulation↗
PubMed2001

Similarities in sunlight-induced mutational spectra of CpG-methylated transgenes and the p53 gene in skin cancer point to an important role of 5-methylcytosine residues in solar UV mutagenesis.

In the p53 gene of human sunlight-associated skin cancers, 35 % of the mutations involve trinucleotide sequences with the rare base 5-methylcytosine (5'PymCG). In order to determine the involvement of 5-methylcytosine in sunlight-induced mutations, we have analyzed the cII transgene in mouse cells, a mutational target gene that we found is methylated at most CpG sequences. We report that the mutational spectra produced by irradiation with 254 nm UVC radiation and simulated sunlight, respectively, differ most dramatically by the much higher involvement of dipyrimidine structures containing 5-methylcytosine in the solar UV mutation spectrum (32 % versus 9 % of all mutations). A distinct mutational hotspot induced by simulated sunlight occurs at a sequence 5'TmCG and is associated with high levels of cis-syn cyclobutane pyrimidine dimer formation. A comparison of sunlight-induced mutational spectra of the cII and lacI transgenes, as well as the p53 gene in skin tumors, shows that 5-methylcytosine is involved in 25 to 40 % of all mutations in all three systems. The combined data make a strong case that cyclobutane pyrimidine dimers forming preferentially at dipyrimidine sequences with 5-methylcytosine are responsible for a considerable fraction of the mutations induced by sunlight in mammalian cells.

5-Methylcytosine↗
PubMed1998

Bacterial expression and characterization of chicken apolipoprotein A-I.

Apolipoprotein (apo) A-I is a 28-kDa exchangeable apolipoprotein that plays a key role in lipoprotein metabolism. It is widely distributed among animal species and is rich in alpha-helical secondary structure. Unlike human apoA-I, which aggregates in the absence of lipid, chicken apoA-I is monomeric in the lipid-free state. To take advantage of this physical characteristic, a bacterial expression system for production of recombinant chicken apoA-I has been developed. The cDNA-encoding chicken apoA-I was cloned into the pET expression vector under the regulation of the lac operon and transformed into Escherichia coli. Recombinant apoA-I protein recovered from the soluble fraction of the bacterial cell pellet was purified to greater than 95% homogeneity by reversed-phase high-performance liquid chromatography. Although immunoblot analysis confirmed the identity of the overexpressed protein, its migration on denaturing polyacrylamide gel electrophoresis was slower than its natural counterpart. To determine if the vector-encoded 18 residue pelB N-terminal leader sequence was not cleaved by the bacterial leader peptidase, isolated recombinant chicken apoA-I was incubated with exogenous leader peptidase. This treatment resulted in an increased electrophoretic mobility, with migration to a position corresponding to plasma-derived chicken apoA-I. Electrospray mass spectrometry indicated a mass of 27,961 +/- 4 Da, in agreement with that predicted for natural chicken apoA-I. Far-UV circular dichroism spectroscopy indicated an alpha-helical content similar to apoA-I isolated from chicken plasma, suggesting that the protein is folded in solution. Fluorescence studies showed that the wavelength of maximum fluorescence emission of the two tryptophan residues in the protein was 331 nm, with no shift occurring following complexation with lipid. Recombinant apoA-I was shown to be functional in lipoprotein binding as well as to possess an ability to transform bilayer vesicles of dimyristoylphosphatidylcholine into discoidal complexes. This is the first report of bacterial expression of an avian apoA-I. Increased availability and the potential for site-directed mutagenesis of this protein will aid in further characterization of apoA-I and the mechanism whereby it functions in cholesterol transport.

Animals↗
PubMed2001

Copurification of the Lac repressor with polyhistidine-tagged proteins in immobilized metal affinity chromatography.

One of the commonly used resins for immobilized metal affinity purification of polyhistidine-tagged recombinant proteins is TALON resin, a cobalt (II)--carboxymethylaspartate-based matrix linked to Sepharose CL-6B. Here, we show that TALON resin efficiently purifies the native form of Lac repressor, which represents the major contaminant when (His)(6)-tagged proteins are isolated from Escherichia coli host cells carrying the lacI(q) gene. Inspection of the crystal structure of the repressor suggests that three His residues (residues 163, 173, and 202) in each subunit of the tetramer are optimally spaced on an exposed face of the protein to allow interaction with Co(II). In addition to establishing a more efficient procedure for purification of the Lac repressor, these studies indicate that non-lacI(q)-based expression systems yield significantly purer preparations of recombinant polyhistidine-tagged proteins.

Aspartic Acid↗
PubMed1986

Differential expression of SOS genes in an E. coli mutant producing unstable lexA protein enhances excision repair but inhibits mutagenesis.

The lexA41 mutant of E. coli is a UV-resistant derivative of another mutant, lexA3, which produces a repressor that is not cleaved following inducing treatments. lexA41 carried an additional mutation which changed amino acid 132 in the LexA protein from Ala to Thr. The resultant protein was unstable and was degraded both before and after an inducing treatment. This instability was greater at 42 degrees than at 30 degrees. The protein was more stable in Lon- mutants at both temperatures. lac operon fusions to most of the genes in the SOS regulon were used to show that the various damage-inducible genes were derepressed to different extents. uvrA, B, and D were almost fully derepressed. Consistant with this finding, the rate of removal of T4 endonuclease V-sensitive sites was more rapid in the UV-irradiated lexA41 mutant than in normal cells, suggesting a more active excision repair system. We propose that the instability of the LexA41 protein reduces the intracellular concentration of repressor to a level that allows a high level of excision repair. The additional observation that SOS mutagenesis was only weakly induced in a lexA41 uvrA- mutant implies that the mutant protein partially represses one or more genes whose products promote SOS mutagenesis.

Alleles↗
PubMed1977

Transposition and insertion of intact, deleted and enlarged ampicillin transposon Tn3 from mini-R1 (Rsc) plasmids into transfer factors.

The miniR1-(Rsc)-plasmids which derive from the copy mutant R1drd-19B2 (pKN102) are non-conjugative extrachromosomal elements which can not be co-transferred by various transfer factors to recipient strains under standard mating conditions. The attempts to mobilize Rsc11 by F'lac lead to transconjugants carrying F'lac::Tn3 with Tn3 mainly inserted into the lac operon. In addition it can be shown that Rsc11 can become inserted as a complete unit into the transfer factor giving rise to rather unstable recombinant intermediates. Dissociation of these intermediates may lead to alterations of the original plasmids. The Tn3 part of Rsc13 can be enlarged or deleted by in vitro manipulations. In vitro insertion of EcoRI-fragments into an EcoRI+ site of Tn3 leads to new transposable units which can be transposed to the RTF part of R1. This new genetic entity can be stably integrated into the chromosome of E. coli by integrative suppression of a dnaAts-mutation. Deletions at one end or the central region of Tn3 abolish the capability of transposition. However, the Rsc-plasmids containing the deleted Tn3 can still be inserted into the transfer factor as complete units. The resulting recombinants are unstable leading after dissociation in some cases to new plasmids with altered properties.

Ampicillin↗
PubMed1978

Translation and mRNA decay.

Degradation of messenger RNA from the lactose operon (lac mRNA) was measured during the inhibition of protein synthesis by chloramphenicol (CM) or of translation-initiation by kasugamycin (KAS). With increasing CM concentration mRNA decay becomes slower, but there is no direct proportionality between rates of chemical decay and polypeptide synthesis. During exponential growth lac mRNA is cleaved endonucleolytically (Blundell and Kennell, 1974). At a CM concentration which completely inhibits all polypeptide synthesis this cleavage is blocked. In contrast, if only the initiation of translation is blocked by addition of KAS, the cleavage rate as well as the rate of chemical decay are increased significantly without delay. These faster rates do not result from immediate degradation of the lengthening stretch of ribosome-free proximal message, since the full-length size is present and the same discrete message sizes are generated during inhibition. These results suggest that neither ribosomes nor translation play an active role in the degradative process. Rather, targets can be protected by the proximity of a ribosome, and without nearly ribosomes the probability of cleavage becomes very high. During normal growth there is a certain probability that any message is in such a vulnerable state, and the fraction of vulnerable molecules determines the inactivation rate of that species.

Aminoglycosides↗
PubMed1975

Catabolite repression in Escherichia coli K12 mutants defective in glucose transport.

The phenomenon of glucose catabolite repression was studied in Escherichia coli mutants unable to transport this carbohydrate. The pts I,H mutant P34 was much less sensitive to permanent and transient repressive effect of glucose on beta-galactosidase synthesis than parental type. The 1103 mutant with lack of enzyme 1 of the phosphoenolpyruvate-dependent phosphotransferase system (ptsI) behaves as well as P34 mutant after addition of glucose to casamino acids mineral medium. But in minimal medium with succinate as the sole source of carbon cells of the 1103 mutant (in accordance with the data of Perlman and Pastan, 1969) show hightened sensibility to transient glucose repression. The effect of hypersensibility disappears when the lacI mutation rendering the beta-galactosidase synthesis to costitutivity is introduced in 1103 mutant. It is shown that the hightened sensibility of beta-galactosidase synthesis to glucose transient repression in 1103 mutant is not an effect of the pts mutation and most probably is due to "inducer exclusion" of the lac operon. It is also shown that if one introduces the P34 mutation in strain devoided of one of the enzymes II for glucose (gptA) (and due to this resistant to glucose catabolite repression) then the level of resistance in double mutant does not increase in spite of considerable supression of 14C glucose accumulation. It is discussed the role of separate components of Escherichia coli K12 glucose transport system in realization of the phenomenon of catabolite repression.

Biological Transport↗
PubMed1989

Characterization of phosphoenolpyruvate synthase mutants in Salmonella typhimurium.

The enteric bacteria are able to grow by utilizing three-carbon compounds (pyruvate, lactate, and alanine) as sole carbon sources only if they have a functional phosphoenolpyruvate synthase (PEP synthase). PEP synthase catalyzes the phosphorylation of pyruvate to PEP with the hydrolysis of ATP to AMP. This anaplerotic reaction is needed for the synthesis of carbohydrates and citric acid cycle intermediates that are essential for continued cell growth. Insertion mutants were isolated in Salmonella typhimurium that specifically lack the ability to grow on three-carbon compounds. These mutants also fail to utilize acetate as a sole carbon source. Enzyme assays were performed and the results showed that these mutants contain no PEP synthase activity. By using bacteriophage P22, the pps mutations isolated in this study were found to be contransducible with genetic markers in both the aroD and btuC genes. Three-factor crosses pinpointed the order of these genes and their distances with respect to each other. One of the mutants carries a pps::lac operon fusion. This fusion was used to explore the transcriptional regulation of the pps gene. A functional copy of the pps gene is required for its own induction. The pps gene is also under catabolite repression, but the addition of adenosine 3',5'-cyclic monophosphate (cyclic AMP) to cells grown in the presence of glucose does not relieve this repression. These results indicate that the synthesis of PEP synthase is regulated in a more complex manner than has been previously recognized.

Cyclic AMP↗
PubMed1987

Regulation of the fdhF gene encoding the selenopolypeptide for benzyl viologen-linked formate dehydrogenase in Escherichia coli.

Two classes of mutants defective in benzyl viologen-linked formate dehydrogenase (FDH-BV) activity were isolated from Escherichia coli K12. Class I consisted of four mutants which were specifically devoid of FDH-BV activity. Their mutation mapped between the ssb and melA genes at 92 min on the genome, at a site recently designated fdhF by Pecher et al. (1985). The direction of transcription of gene fdhF was found to be counterclockwise on the E. coli chromosome in one Mudl(Aprlac) fusion mutant. Expression of the lac operon in this mutant was induced by formate and repressed by nitrate, nitrite or trimethylamine N-oxide. It was found to be dependent on the positive control exerted by the fdhA, B and C genes, possibly involved in selenium incorporation, and by an hydB gene affecting the formate hydrogenlyase pathway. Class II, represented by one Mudl(Aprlac) mutant, exhibited no FDH-BV activity and a reduced level of hydrogenase activity. The relevant fdv mutation was shown to be located at 58 min and to affect the expression of fdhF.

Aldehyde Oxidoreductases↗
PubMed1979

Relief of polarity in E. coli depleted of 30S ribosomal subunits.

Escherichia coli was depleted of ribosomes by a thermal shock at 47 degrees C which quantitatively destroyed the 30S ribosomal subunits. During recovery in minimal medium at 30 degrees C RNA is synthesized while protein synthesis resumes only after about 90 min. It is shown that lac mRNA is synthesized in the complete absence of ribosomal activity and hence RNA synthesis is not coupled to protein synthesis. Lac mRNA from a series of lac nonsense mutants was examined in both heated and untreated cells. It was found that the polar effect of nonsense mutation is relieved in the absence of ribosomes and that this relief is due to the synthesis of larger mRNA molecules. Since Rho remained active in thermally treated cells, premature termination at secondary signals within the lac operon must also depend on the presence of active ribosomes.

Escherichia coli↗
PubMed1988

Functional and structural homology among regulatory cistrons of pili-adhesin determinants in Escherichia coli.

Expression of the digalactoside-binding Pap pili involves two trans-acting regulatory genes, papB and papI. Using pap-lac operon fusions and DNA hybridization probes derived from pap DNA we tested whether or not other pili-adhesin determinants from different Escherichia coli strains encode homologs to the pap regulatory genes. Digalactoside-specific clones of serotypes F72 and F11 complemented papB and papI mutants of the Pap (serotype F13) clone and DNA hybridization analysis showed that the clones are homologous in the DNA sequences encoding the two regulatory genes. Similar results were obtained with an S-pili determinant which mediates binding to sialic acid-containing receptors and the findings suggest that the regulatory regions may be more conserved than other genes in different pili-adhesin gene clusters. Determinants for type 1-pili (mannose-specific binding) and for pili associated with enterotoxigenic E. coli (K88, K99, CFAI, CFAII) did not appear to contain DNA sequences homologous to papB or papI. E. coli strain J96, which was the origin of the pap DNA, was found to carry two additional copies of papB-papI homologous sequences in the chromosome. In strains expressing more than one kind of pili the trans-active gene products thereby may allow for regulatory interaction between separate pili-adhesin gene systems.

Base Sequence↗
PubMed1983

Identification of an iron uptake system specific for coprogen and rhodotorulic acid in Escherichia coli K12.

With the lac operon fusion technique, mutants were isolated in two genes that specify two outer membrane proteins designated FhuE (76 K) and Fiu (83 K). The synthesis of both proteins was increased under low iron growth conditions. The FhuE-protein was shown to be necessary for iron uptake via coprogen, an iron chelator produced by certain fungi, e.g. Neurospora crassa. In addition to fhueE the genes fhuCDB, tonB and exbB were necessary for iron coprogen uptake. The gene fhuE was mapped between kdp and gltA near 16 min on the genetic map of E. coli K12, while gene fiu was mapped near 18 min between chlA and chlE. Nor iron transport system could be assigned as yet to the Fiu protein.

Bacterial Outer Membrane Proteins↗
PubMed1982

Only one gene is required for the glpT-dependent transport of sn-glycerol-3-phosphate in Escherichia coli.

Deletion and point mutants defective in the glpT-dependent sn-glycerol-3-phosphate transport system were isolated and located on the Escherichia coli chromosome. They mapped in glpT in the clockwise order gyrA, glpA, glpT at around 48 min on the Escherichia coli linkage map. The mutations within glpT were ordered by deletion mapping, three factor crosses, and by crosses involving lambda transducing bacteriophages carrying glpT-lac operon fusions. Results obtained using these fusion phages indicated that glpT is transcribed in the counterclockwise direction on the E. coli linkage map. Complementation analysis using these mutants revealed only one complementation group. Thus, one gene is necessary and sufficient for the proton motive force-dependent sn-glycerol-3-phosphate transport system.

Biological Transport↗
PubMed1996

Selectively 13C-enriched DNA: evidence from 13C1' relaxation rate measurements of an internal dynamics sequence effect in the lac operator.

In order to study some internal dynamic processes of the lac operator sequence, the 13C-labeled duplex 5'd(C0G1C2T3C4A5C6A7A8T9T10).d(A10A9T8T7G6T5G4A3G2C1G0)3' was used. The spreading of both the 1H1' and 13C1' resonances brought about an excellent dispersion of the 1H1'-13C1' correlations. The spin-lattice relaxation parameters R(Cz), R(Cx,y) and R(Hz --> Cz) were measured for each residue of the two complementary strands, except for the 3'-terminal residues which were not labeled. Variation of the relaxation rates was found along the sequence. These data were analyzed in the context of the model-free formalism proposed by Lipari and Szabo [(1982) J. Am. Chem. Soc., 104, 4546-4570] and extended to three parameters by Clore et al. [(1990) Biochemistry, 29, 7387-7401; and (1990) J. Am. Chem. Soc., 112, 4989-4991]. A careful analysis using a least-squares program showed that our data must be interpreted in terms of a three-parameter spectral density function. With this approach, the global correlation time was found to be the same for each residue. All the C1'-H1' fragments exhibited both slow (tau s = 1.5 ns) and fast (tau f = 20 ps) restricted libration motions (Ss2 = 0.74 to 1.0 and Sf2 = 0.52 to 0.96). Relaxation processes were described as governed by the motion of the sugar relative to the base and in terms of bending of the whole duplex. The possible role played by the special structure of the AATT sequence is discussed. No evident correlation was found between the amplitude motions of the complementary residues. The 5'-terminal residues showed large internal motions (S2 = 0.5), which describe the fraying of the double helix. Global examination of the microdynamical parameters Sf2 and Ss2 along the nucleotide sequence showed that the adenine residues exhibit more restricted fast internal motions (Sf2 = 0.88 to 0.96) than the others, whereas the measured relaxation rates of the four nucleosides in solution were mainly of dipolar origin. Moreover, the fit of both R(Cz) and R(Hz --> Cz) experimental relaxation rates using an only global correlation time for all the residues, gave evidence of a supplementary relaxation pathway affecting R(Cx,y) for the purine residues in the (5' --> 3') G4A3 and A10A9T8T7 sequences. This relaxation process was analyzed in terms of exchange stemming from motions of the sugar around the glycosidic bond on the millisecond time scale. It should be pointed out that these residues gave evidence of close contacts with the protein in the complex with the lac operator [Boelens et al. (1987) J. Mol. Biol., 193, 213-216] and that these motions could be implied in the lac-operator-lac-repressor recognition process.

Bacterial Proteins↗
PubMed1981

A mutant rho ATPase from Escherichia coli that is temperature-sensitive in the presence of RNA.

The Escherichia coli mutant rho-115 suppresses lac operon polarity conferred by the lacZ::IS1 insertion MS319. The ATPase activity of purified rho-115 protein was maximal at 40 degrees C, in contrast to 45 degrees C for rho+. At higher temperatures (50 degrees C, 55 degrees C), the fractions of activities at maximal temperature were consistently lower for rho-115 compared to rho+. The 30-minute time course of rho-115 ATP hydrolysis was linear at 37 degrees C but at 45 degrees C the linear kinetics of hydrolysis reached a plateau between 10 and 15 minutes. The 30-minute time courses for rho+ were linear at both 37 degrees C and 45 degrees C. The rho-115 and rho+ ATPase activities were equally heat-stable during preincubation at 45 degrees C in buffer. Inclusion of ATP during preincubation protected these rho proteins from inactivation to the same extent. The presence of polyC during preincubation protected rho+ activity but produced substantial inactivation of rho-115 ATPase. The presence of polyU during preincubation gave similar results. Concentrations of polyC between 625 ng/ml and 100 micrograms/ml yielded the same extent of rho-115 ATPase inactivation during preincubation at 45 degrees C. Thermal inactivation of rho-115 ATPase by polyC was halted by shifting preincubation temperature from 45 degrees C to 35 degrees C, indicating that polyC-induced destabilization of rho-115 was irreversible.

Adenosine Triphosphatases↗
PubMed1987

Separate regulatory systems for the repression of metE and btuB by vitamin B12 in Escherichia coli.

Synthesis of the btuB-encoded outer membrane receptor for vitamin B12 and the metE-encoded homocysteine methyltransferase is repressed by growth of Escherichia coli in the presence of vitamin B12. The regulation by vitamin B12 of the production of beta-galactosidase in strains carrying btuB-lac or metE-lac operon fusions indicated that repression of both genes operates at the transcriptional level. Selection for expression of these fusions under repressive conditions allowed isolation of second-site mutations in which repressibility by vitamin B12 had been lost. Mutations in metH and metF prevented vitamin B12-dependent regulation of metE, but not that of btuB. Mutations in btuB and other genes involved in uptake of the vitamin eliminated or reduced repression. Mutations in the newly identified gene, btuR, controlled the repressibility of btuB, but had no effect on metE regulation. The btuR gene resides at 27.9 min on the genetic map in the gene order cysB-topA-btuR-trp; it acts in a trans-dominant manner and appears to encode a repressor of btuB transcription.

Bacterial Outer Membrane Proteins↗