Mutations partially inactivating the lactose repressor of Escherichia coli

After treatment with N-methyl-N'-nitro-N-nitrosoguanidine, 133 independent mutants of a haploid strain of Escherichia coli able to use phenyl-beta-galactoside as a carbon source were obtained. The galactoside was specific in selecting for mutants with increases in their uninduced levels of beta-galactosidase. Virtually all mutants (37 in a subsample of 38) carried mutations in the lac repressor gene. There were two classes of repressor mutants. As well as the commonly identified class of mutants with completely inactivated repressors, there was a frequent class of mutants (21/37) whose repressors were partially inactivated. Most of these (15/21) repressed beta-galactosidase synthesis 4 to 50 times less than wild type, but were more numerous in the lower part of this range. Their beta-galactosidase was inducible to levels characteristic of the parent strain. The repressor activities were diverse and stably expressed under routine growth conditions. The decreased activity did not result from the formation of temperature-sensitive repressors. None of the mutants with completely inactivated repressors appeared to carry UAG or UGA chain-terminating codons. On the assumption that the partially defective repressor mutants carried missense mutations, it is argued that missense mutations in the lac repressor gene modify the repressor's affinity for the operator with high probability. An explanation is proposed for the apparent sensitivity of this repressor function to partial inactivation as the result of amino acid substitutions.     

Comparing native and irradiated E. coli lactose repressor-operator complex by molecular dynamics simulation

The function of the E. coli lactose operon requires the binding of the tetrameric repressor protein to the operator DNA. We have previously shown that γ-irradiation destabilises the repressor-operator complex because the repressor gradually loses its DNA-binding ability (Radiat Res 170:604–612, 2008). It was suggested that the observed oxidation of tyrosine residues and the concomitant structural changes of irradiated headpieces (DNA-binding domains of repressor monomers) could be responsible for the inactivation. To unravel the mechanisms that lead to repressor-operator complex destabilisation when tyrosine oxidation occurs, we have compared by molecular dynamic simulations two complexes: (1) the native complex formed by two headpieces and the operator DNA, and (2) the damaged complex, in which all tyrosines are replaced by their oxidation product 3,4-dihydroxyphenylalanine (DOPA). On a 20 ns time scale, MD results show effects consistent with complex destabilisation: increased flexibility, increased DNA bending, modification of the hydrogen bond network, and decrease of the positive electrostatic potential at the protein surface and of the global energy of DNA-protein interactions.     

Radiation-induced oxidative damage to the DNA-binding domain of the lactose repressor

Understanding the cellular effects of radiation-induced oxidation requires the unravelling of key molecular events, particularly damage to proteins with important cellular functions. The Escherichia coli lactose operon is a classical model of gene regulation systems. Its functional mechanism involves the specific binding of a protein, the repressor, to a specific DNA sequence, the operator. We have shown previously that upon irradiation with gamma-rays in solution, the repressor loses its ability to bind the operator. Water radiolysis generates hydroxyl radicals (OH* radicals) which attack the protein. Damage of the repressor DNA-binding domain, called the headpiece, is most likely to be responsible of this loss of function. Using CD, fluorescence spectroscopy and a combination of proteolytic cleavage with MS, we have examined the state of the irradiated headpiece. CD measurements revealed a dose-dependent conformational change involving metastable intermediate states. Fluorescence measurements showed a gradual degradation of tyrosine residues. MS was used to count the number of oxidations in different regions of the headpiece and to narrow down the parts of the sequence bearing oxidized residues. By calculating the relative probabilities of reaction of each amino acid with OH. radicals, we can predict the most probable oxidation targets. By comparing the experimental results with the predictions we conclude that Tyr7, Tyr12, Tyr17, Met42 and Tyr47 are the most likely hotspots of oxidation. The loss of repressor function is thus correlated with chemical modifications and conformational changes of the headpiece.     

Differential binding of allolactose anomers to the lactose repressor of Escherichia coli.

Preferential binding of the β-anomer of allolactose to the lactose repressor of Escherichia coli was demonstrated by two methods: (1) by repeated washing of ammonium sulfate precipitates of the allolactose-repressor complex and (2) by competitive inhibition of allolactose binding by isopropyl-β-d-thiogalacto-side. Quantitation showed that one β-allolactose binds per isopropyl-β-d-thiogalactoside binding site. A control system is postulated.     

Site-directed mutagenesis of lysine 319 in the lactose permease of Escherichia coli.

Lys319, which is on the same face of putative helix X as His322 and Glu325 in the lactose permease of Escherichia coli, has been replaced with Leu by oligonucleotide-directed, site-specific mutagenesis. Although previous experiments suggested that the mutation does not alter permease activity, we report here that K319L permease is unable to catalyze active lactose accumulation or lactose efflux down a concentration gradient. The mutant does catalyze facilitated influx down a concentration gradient at a significant rate; however, the reaction occurs without concomitant H+ translocation. The mutant also catalyzes equilibrium exchange at about 50% of the wild-type rate, but it exhibits poor counterflow activity. Finally, flow dialysis and photoaffinity labeling experiments with p-nitrophenyl alpha-D-galactopyranoside indicate that K319L permease probably has a markedly decreased affinity for substrate. The alterations described are not due to diminished levels of the mutated protein in the membrane, since immunological studies reveal comparable amounts of permease in wild-type and K319L membranes. It is proposed that Lys319, like Arg302, His322, and Glu325, plays an important role in active lactose transport, as well as substrate recognition.     

Ultraviolet radiation-induced derepression of the lactose operon of E. coli

The effect of ultraviolet irradiation of a regulatory protein, the lac repressor, on its interactions with operator DNA is investigated by spectroscopic and electrophoresis methods. A second set of experiments is performed to assay the capacity of the system containing the irradiated repressor to be induced by IPTG. The protein-nucleic acid interactions are modified upon ultraviolet irradiation of the repressor. The inducer becomes ineffective and repressor stays "locked" to DNA in conditions in which the native repressor is released from the system. These facts are discussed in terms of genes repression and of promotion step in ultraviolet induced carcinogenesis.     

Modification of tyrosine residues of the lactose repressor protein.

Reaction of the lactose repressor protein from Escherichia coli with high molar excesses (up to 800 fold) of tetranitromethane resulted in modification of tyrosine residues in the amino-terminal and core regions of the molecule. Tyrosines 7 and 17 exhibit significant reactivity at low levels (5-10 fold molar excess) of tetranitromethane. The loss of operator binding activity upon nitration at these low concentrations of reagent indicates involvement of these two tyrosines in the binding process. Inducer binding activity was maintained at approx. 90% of unreacted repressor for all excesses of reagent studied. Addition of inducer to the repressor prior to reaction resulted in decreased modification of tyrosines in the core region, but anti-inducers did not affect the reaction significantly. The effect of inducers on the pattern of reaction apparently reflects the conformational change which occurs upon binding of these ligands. Acetylation of the repressor protein with N-acetylimidazole modified lysines and tyrosines with complete loss of operator binding activity and retention of 75-80% of inducer binding activity.     

Genetic separation of Escherichia coli recA functions for SOS mutagenesis and repressor cleavage.

Evidence is presented that recA functions which promote the SOS functions of mutagenesis, LexA protein proteolysis, and lambda cI repressor proteolysis are each genetically separable from the others. This separation was observed in recombination-proficient recA mutants and rec+ (F' recA56) heterodiploids. recA430, recA433, and recA435 mutants and recA+ (F' recA56) heterodiploids were inducible for only one or two of the three functions and defective for mutagenesis. recA80 and recA432 mutants were constitutively activated for two of the three functions in that these mutants did not have to be induced to express the functions. We propose that binding of RecA protein to damaged DNA and subsequent interaction with small inducer molecules gives rise to conformational changes in RecA protein. These changes promote surface-surface interactions with other target proteins, such as cI and LexA proteins. By this model, the recA mutants are likely to have incorrect amino acids substituted as sites in the RecA protein structure which affect surface regions required for protein-protein interactions. The constitutively activated mutants could likewise insert altered amino acids at sites in RecA which are involved in the activation of RecA protein by binding small molecules or polynucleotides which metabolically regulate RecA protein.     

Enhancement by cysteamine of N-methyl-N-nitrosourea mutagenesis in E. coli

Cysteamine (MEA) is comutagenic to methylnitrosourea (MNU) in E. coli AB 1157 but not in the nonadaptable mutant derivative ada-6 of that strain. The comutagenic action of MEA was eliminated by cysteine at low concentrations, which also lowered mutation frequencies in AB1157 but not in ada-6. In model experiments it was shown that cysteine counteracted the inhibition by MEA of beta-galactosidase induction in both bacterium strains. The comutagenic action of MEA is interpreted as being due to an inhibition of induction of methyltransferase during treatment with MNU.     

Binding of E.coli lac repressor to non-operator DNA*

It is shown by melting profile analysis of lac repressor-DNA complexes that repressor binds tightly and preferentially (relative to single-stranded DNA) to double-stranded non-operator DNA. This binding stabilizes the DNA against melting and the repressor against thermal denaturation. Analysis of the extent of stabilization and the rate of dissociation of repressor from non-operator DNA as a function of sodium ion concentration shows, in confirmation of other studies,(3,4) that the binding constant (K(RD)) is very ionic strength dependent; K(RD) increases from approximately 10(6) M(-1) at approximately 0.1 M Na(+) to values in excess of 10(10) M(-1) at 0.002 M Na(+). Repressor bound to non-operator DNA is not further stabilized against thermal denaturation by inducer binding, indicating that the inducer and DNA binding sites probably represent separately stabilized local conformations. Transfer melting experiments are used to measure the rate of dissociation of repressor from operator DNA. These experiments show that most of the ionic strength dependence of the binding constant is in the dissociation process; the estimated dissociation rate constant decreases from greater than 10(-1) sec(-1) at [Na(+)] >/= 0.02 M to less than 10(-4) sec(-1) at [Na(+)] 相似文献   

Time resolved spectroscopy of the tryptophyl fluorescence of the E. coli LAC repressor.

A new crystal form of a mitogenic lectin from pea seeds ($\text{Pisum sativum}$) has been obtained which is suitable for high resolution structural work. The crystals are orthorhombic, space group P2₁2₁2₁, with unit cell dimensions: $\text{a}$ = 64.2Å, $\text{b}$ = 72. 7Å, $\text{c}$ = 108. 3Å. The asymmetric unit contains one protein molecule.     

In vitro interaction of the Escherichia coli cyclic AMP receptor protein with the lactose repressor

Sedimentation equilibrium studies show that the Escherichia coli cyclic AMP receptor protein (CAP) and lactose repressor associate to form a 2:1 complex in vitro. This is, to our knowledge, the first demonstration of a direct interaction of these proteins in the absence of DNA. No 1:1 complex was detected over a wide range of CAP concentrations, suggesting that binding is highly cooperative. Complex formation is stimulated by cAMP, with a net uptake of 1 equivalent of cAMP per molecule of CAP bound. Substitution of the dimeric lacI-18 mutant repressor for tetrameric wild-type repressor completely eliminates detectable binding. We therefore propose that CAP binds the cleft between dimeric units in the repressor tetramer. CAP-lac repressor interactions may play important roles in regulatory events that take place at overlapping CAP and repressor binding sites in the lactose promoter.     

E. coli F1-ATPase: site-directed mutagenesis of the beta-subunit

Residues beta Glu-181 and beta Glu-192 of E. coli F1-ATPase (the DCCD-reactive residues) were mutated to Gln. Purified beta Gln-181 F1 showed 7-fold impairment of 'unisite' Pi formation from ATP and a large decrease in affinity for ATP. Thus the beta-181 carboxyl group in normal F1 significantly contributes to catalytic site properties. Also, positive catalytic site cooperativity was attenuated from 5 X 10(4)- to 548-fold in beta Gln-181 F1. In contrast, purified beta Gln-192 F1 showed only 6-fold reduction in 'multisite' ATPase activity. Residues beta Gly-149 and beta Gly-154 were mutated to Ile singly and in combination. These mutations, affecting residues which are strongly conserved in nucleotide-binding proteins, were chosen to hinder conformational motion in a putative 'flexible loop' in beta-subunit. Impairment of purified F1-ATPase ranged from 5 to 61%, with the double mutant F1 less impaired than either single mutant. F1 preparations containing beta Ile-154 showed 2-fold activation after release from membranes, suggesting association with F0 restrained turnover on F1 in these mutants.     

An approach to analyzing UV mutagenesis in E. coli

UV mutagenesis of single-strand DNA phage can be divided into three types: induced untargeted; induced targeted; and uninduced targeted. We report the development of new tools to determine the number of processes which contribute to these types of mutagenesis. An E. coli tRNA gene, glyU, has been cloned using M13 derivatives mp8 and mp9 as vectors. The nucleotide sequence of glyU and its flanking regions is presented. In this paper, phage glyU anticodon mutants are detected by their ability to suppress GAA and GAT missense mutations in trpA. We used phage carrying GAG and CTC at the anticodon position and found results consistent with the hypothesis that two processes act to produce the transition to GAA suppression: an uninduced regionally targeted process; and an induced locally targeted process with some untargeted activity. The transversion frequency to GAT suppression on the other hand responded as if only an uninduced locally targeted process was involved. Thus, we hypothesize that the new tools have discriminated three different processes of mutagenesis and we discuss further work designed to test this hypothesis.