lac repressor-operator interaction. Analysis of the X86 repressor mutant

In vitro measurements show that the X86 repressor, which has an increased affinity for the lac operator as compared to wild-type repressor, also has an increased affinity for non-operator sites on Escherichia coli DNA. The rate constant of association of repressor and operator is decreased by E. coli DNA fivefold more for X86 repressor than for wild-type repressor. Low inducer concentrations increase the rate of association of X86 repressor and operator in the presence of E. coli DNA. In a partial equilibrium situation where part of the X86 repressor is bound to the operator, and part to either non-operator sites on E. coli DNA or to an O^c operator, the formation of complexes between X86 repressor and wild-type operator is favored by low inducer concentrations. Repression of the lac enzymes increases drastically in the X86 mutant in the absence of DNA synthesis in vivo. A new explanation for the in vivo characteristics of the X86 mutant is suggested.     

Cellular levels of the prophage lambda and 434 repressors.

As a prerequisite to a quantitative study of the inactivation of phage repressors in vivo (Bailone et al., 1979), the cellular concentrations of the bacteriophage λ and 434 repressors have been measured in bacteria with varying repressor levels.Using the DNA-binding assay we have determined the conditions for optimal repressor titration. The sensitivity of the λ repressor assay was increased by adding magnesium ions to the binding mixture; this procedure was without effect on the titration of the 434 repressor. The measures of the cellular repressor concentrations varied with the method of cell disruption.The cellular concentration of λ repressor, about 140 active repressor molecules per monolysogen, was relatively constant under specific cultural conditions. The repressor concentration increased with the number of cI gene copies but not in direct proportion.The 434 repressor concentration, hardly detectable in extracts of lysogens carrying an imm434 prophage, was greatly enhanced in bacteria carrying the newly constructed plasmid pGY101, that encodes the 434 cI gene.The cellular repressor level produced by 434 is lower than that produced by λ: this indicates that the maintenance of the prophage state is ensured by a relatively small number of repressor molecules binding tightly to the operator sites.     

Direct identification of the amino acid changes in two mutant lac repressors.

Deletions extending into the trp operon at one terminus and the lacI control region at the other terminus have been examined. One of these, B116, ends within the trp leader sequence and eliminates the trp attenuator site, placing the synthesis of lac repressor under trp control. We have isolated and characterized the B116 repressor. The protein sequence of the aminoterminus of B116 shows that an additional 16 residues are added to the amino-terminal end of wild-type repressor. Moreover, a valine residue appears in place of methionine at position 17 (the original amino-terminal residue of the wild-type repressor). A comparison of the messenger RNA sequence of the trp leader region and of the I leader region demonstrates that the translation of the B116 repressor is initiated at an AUG codon within the trp leader sequence. The GUG initiation codon at the start point for translation of wild-type repressor is now read as valine, since it appears at an internal position (residue 17 of the altered repressor). The B116 repressor accumulates at levels as high as 1% of the soluble cell protein in trpR^? strains. The efficiency of the trp leader initiation codon in translation suggests that in wild-type strains this AUG is also active in directing protein synthesis, which would result in a polypeptide consisting of 14 amino acids. We have examined the physical properties of the B116 repressor, which shows a marked tendency to form higher aggregates. Other characteristics of B116 are also described.     

Proposed alpha-helical super-secondary structure associated with protein-dna recognition

Knowledge of the three-dimensional structure of the bacteriophage λ Cro repressor, combined with an analysis of amino acid sequences and DNA coding sequences for this and other proteins that recognize and bind specific base sequences of double-helical DNA, suggests that a portion of the structure of the Cro repressor that is involved in DNA binding also occurs in the Cro protein from bacteriophage 434, the cII protein from bacteriophage λ, the Salmonella phage P22 c2 repressor and the cI repressor from bacteriophage λ. This α-helical super-secondary structure may be a common structural motif in proteins that bind double-helical DNA in a base sequence-specific manner.     

Inactivation of prophage lambda repressor in vivo.

Jacob &; Monod (1961) postulated that prophage A induction results from the inactivation of the λ repressor by a cellular inducer. Although it has been shown that the phage A repressor is inactivated by the recA gene product in vitro (Roberts et al., 1978), we wanted to determine the action of the “cellular inducer” in vivo. Our results have led to a new model, which defines the relationship between the “cellular inducer” and the recA gene product.In order to quantitate the action of the cellular inducer on the λ repressor, we made use of bacteria with elevated cellular levels of the λ repressor (hyperimmune lysogens). We determined the kinetics of repressor inactivation promoted by three representative inducing treatments: ultraviolet light irradiation, thymine deprivation and temperature shift-up of tif-1 mutants.The kinetics of repressor decay in wild-type monolysogens indicate that repressor inactivation is a relatively slow cellular process that takes a generation time to reach completion. Incomplete inactivation of the repressor without subsequent prophage development may occur in a cell. We call this phenomenon detected at the biochemical level “subinduction”. In hyperimmune lysogens. subinduction is always the case.A high cellular level of A repressor that prevents prophage λ induction does not prevent induction of a heteroimmune prophage such as 434 or 80. Although the cellular inducer does not seem specific for any inducible prophage, it does not inactivate two prophage repressors present in a cell in a random manner. We have called this finding “preferential repressor inactivation”. Preferential repressor inactivation may be accounted for by considering that the intracellular concentration of a repressor determines its susceptibility to the action of the inducer.In bacteria with varying repressor levels, a fixed amount of repressor molecules is inactivated per unit of time irrespective of the initial repressor concentration. The rate of repressor inactivation depends on the catalytic capacity of the cellular inducer that behaves as a saturated enzyme. In wild-type bacteria the cellular inducer seems to be produced in a limited amount, to have a weak catalytic capacity and a relatively short half-life. The amount of the inducer formed after tif-1 expression is increased in STS bacteria overproducing a tif-1-modified RecA protein. This result is an indication that a modified form of the RecA protein causes repressor inactivation in vivo.From the results obtained we propose a model concerning the formation of the cellular inducer. We postulate that the cellular inducer is formed in a two-step reaction. The is model visualises how the RecA protein can be induced to high cellular concentrations, even though the RecAp protease molecules remain at a low concentration. The latter accounts for the limited proteolytic activity found in vivo.     

Mapping of I gene mutations which lead to repressors with increased affinity for lac operator

A genetic mapping system is used to locate mutations on the lac repressor gene (I) which lead to repressor proteins with an increased affinity for operator DNA. These tight binding repressors (I^tb) are of particular interest since their analysis should allow some conclusions on the mechanism of interaction between repressor and operator. I^tb mutations were found to map in two regions of the I gene. One is near the amino-terminal end, a region which has been shown to be essential for the DNA binding properties of the repressor. The other region in which I^tb mutations were mapped codes for approximately amino acids 255 to 295 of the repressor, a region which had so far not been considered to be essential for the DNA binding properties of the repressor protein.     

Local mobility of the lac repressor molecule

The rotational mobility of lac repressor from Escherichia coli was investigated by nanosecond fluorescence depolarization spectroscopy. A single rotational correlation time (φ) of the repressor was observed by monitoring the emission anisotropic decay of the intrinsic tryptophan fluorescence. The small value of φ (9·5 ns) suggests that one or both of the two tryptophan residues in the repressor are located in a flexible segment of the protein molecule. This segmental flexibility is enhanced by binding of inducer (isopropyl-β-d-thiogalactoside) to the repressor while it is restrained by binding of anti-inducer (glucose) or small DNA fragments, as indicated by the changes in φ. Further time-dependent emission anisotropy studies with an extrinsic fluorescent probe, N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonate, covalently attached to the repressor yielded two rotational correlation times. The shorter φ_S (6·7 ns) also corresponds to a segmental flexibility whereas the longer φ_L (118 ns) represents the rotational motion of the entire repressor molecule. Both the values of φ_S and φ_L vary by addition of inducer or anti-inducer in a manner similar to that observed for the intrinsic tryptophan fluorescence but they are insensitive to addition of DNA fragments. The changes in local mobility of the lac repressor molecule observed in these studies may provide some insight into how inducer (or anti-inducer) destabilizes (or stabilizes) the repressor-operator complex.     

Kinetics of RecA protein-directed inactivation of repressors of phage lambda and phage P22

Escherichia coli recA protein directs the inactivation of the repressor of Salmonella typhimurium phage P22 in vitro. As is true for repressor of the E. coli phage λ, inactivation of P22 repressor is accompanied by proteolytic cleavage of the repressor into two detectable fragments.We have investigated the kinetics of inactivation of the λ and P22 repressors in vitro. The fraction of λ repressor inactivated per unit time decreases as its concentration in the reaction is increased. However, high concentrations of λ repressor do not inhibit the inactivation of P22 repressor. Thus, it does not appear that the inactivation system is saturated by λ repressor, but rather that λ repressor is a less efficient substrate at higher concentrations.     

Heterogeneity of the two tryptophanyl residues in the lac repressor of Escherichia coli.

The wild-type lac repressor of Escherichia coli is a tetrameric protein which contains two tryptophanyl residues per subunit at positions 190 and 209. Solute perturbation studies of the tryptophan fluorescence of the repressor were performed using a polar but uncharged quencher, acrylamide, to prevent possible bias caused by ionic quenchers. The results indicate that the two tryptophan residues have different accessibilities to the quencher. In addition, contrary to a previous report, the accessibility of these tryptophan residues is not altered by isopropyl-β-d-thiogalactoside (IPTG) binding to the repressor. Similar studies with mutant lac repressor containing only a single tryptophan either at positions 190 or 209 suggest that tryptophan 209 is located in a region which is perturbed by inducer binding. That the two tryptophanyl residues have heterogeneous environments was further confirmed by nanosecond fluorescence spectroscopy which showed the wild-type lac repressor exhibiting two excited-state lifetimes, τ₁ = 5.3 ns and τ₂ = 10 ns. In the presence of 10^?3m IPTG, only a single lifetime of 6 ns was observed for the wild-type repressor suggesting that the inducer perturbs the tryptophan residue with the longer lifetime but not the one with the shorter lifetime. This is in accord with the observation that the mutant repressor containing only tryptophan 190 (the Tyr-209 repressor) has a single lifetime of 4.5 ns which is not altered by IPTG binding. The surprising finding that the mutant repressor which contains only tryptophan 209 (the Tyr-190 repressor) shows two excited-state lifetimes has been interpreted to indicate that the repressor either does not exhibit fourfold symmetry in its subunit arrangement or is present in two different conformational states.     

"Second" and "third operator" of the lac operon: an investigation of their role in the regulatory mechanism.

The influence of the two operator-like regions lying within or near the lac regulatory region on the binding of lac repressor to lac operator has been investigated. λdlac phages deleted either for the “second operator” in the beginning of the Z gene or deleted for the “third operator” at the end of the I gene were constructed. In in vitro binding experiments it could be shown that the deletion of secondary repressor binding sites from the lac regulatory region does not significantly alter the stability of the repressor—operator complex. Measuring the rate constant of association of repressor with operator in the presence of a 150-fold excess of unspecific DNA, we observed a concentration-dependent effect of the unspecific DNA, although the ratio of operator to non-operator DNA was kept constant. A small effect of the secondary binding sites is seen on the rate of association of repressor with operator, indicating that the secondary binding sites might play a role in facilitating association of repressor with operator under _{in vivo} conditions.     

lac Repressor-operator interaction. IX. The binding of lac repressor to operators containing Oc mutations

Representative members of the six classes of operator constitutive (O^c) point mutations, which have been mapped and well characterized in vivo, were crossed into λφ80 lac phages. The phage DNAs containing the O^c mutations were used to measure the affinity of the lac repressor (R) for each O^c operator by determining the half-lives of the different RO^c complexes in vitro. The results provide evidence that: (a) the higher the constitutive level of β-galactosidase in vivo, as the result of an O^c mutation, the lower the affinity of the lac repressor for that O^c operator, with a maximum difference of two orders of magnitude in affinity of the repressor for the highest O^c tested as compared to the wild type O⁺ operator; (b) the six classes of O^c operators appear to be twofold degenerate, in that two members of each class, which were previously distinguished by mapping, have the same affinity for the lac repressor; (c) an inducer and an anti-inducer have the same effect on the RO^c complexes as on the RO⁺ complexes; (d) the relationship between induction ratios in vivo and the binding constant of the repressor for each O^c mutation in vitro does not follow the mass action equation but rather a more complex dependency, which is discussed.These results suggest a functional symmetry in the lac operator.     

Lambda phage cro repressor: Non-specific DNA binding

The interaction of lambda phage cro repressor with double-stranded non-specific DNA has been investigated by monitoring the quenching of its intrinsic tyrosyl fluorescence. The McGhee & von Hippel (1974) analysis of the binding of cro repressor to DNA showed that cro repressor undergoes structural variations in the ionic strength range from 0.04 to 0.18m-KCl. Under these salt conditions, the excluded binding site size of cro repressor on the DNA lattice changes from three to four base-pairs (6 to 8 nucleotides) at the lower ionic strengths, to seven to eight base-pairs (14 to 16 nucleotides) at the higher ionic strength. Quaternary structure variation, which does not cause the excluded site size variation, was also noted at low ionic strengths. Evidence is presented to indicate that cro repressor binds only one side of the DNA helix, such that cro repressor covers a stretch of 14 to 16 nucleotides along one side of the helix in the presence of 0.2 m-salt. Under conditions where the cro repressor structure is constant, approximately nine ion-pairs are formed in the cro repressor-non-specific DNA complex. These results are in agreement with the model proposed by Anderson et al. (1981).     

An analysis of repressor overproduction by the lambda cIIIs mutant.

Efficient lysogenization of Escherichia coli K12 by bacteriophage λ requires the high level of synthesis of the phage repressor shortly after infection. This high level of synthesis of repressor requires the action of the λ eII and cIII proteins. Certain mutants of λ (λcIIIs) appear to have excess $\text{c}\text{II}\text{c}\text{III}$ activity and can lysogenize more efficiently than λ⁺. The basis for the enhanced lysogenization is that, while two or more infecting phage are necessary for λ⁺ to lysogenize, a single infecting λcIIIs particle is sufficient for lysogenization. Also, repressor levels in cells infected with λcIIIs are higher than in those infected with λ⁺. I report here that repressor overproduction by λcIIIs (1) is due to a much higher rate of repressor synthesis than that of λ⁺; (2) is most marked at low multiplicities of infection, possibly because λcIIIs produces repressor much more efficiently than λ⁺ as a singly infecting phage.     

Molecular analysis of the malR gene of Clostridium butyricum NCIMB 7423, a member of the LacI-GalR family of repressor proteins

Deletion of a region of DNA 5′ to a previously characterised malQ gene of Clostridium butyricum resulted in increased production of the enzyme activity encoded by malQ, 4-α-glucanotransferase. Nucleotide sequence analysis revealed the presence of an open reading frame capable of encoding a protein of 335 amino acids. This protein was found to share 33% amino acid sequence identity with the Bacillus subtilis CcpA (catabolite control protein) repressor, 28% identity with the Streptomyces coelicolor MalR repressor, and 30%, 25%, and 21% amino acid identity with the Escherichia coli repressors GalR, LacI and MalI, respectively. The amino-terminal domain was predicted to be able to form a helix-turn-helix structure, and shared highest similarity with the equivalent functional domain from the E. coli LacI repressor. Interruption of malR by the generation of a frameshift mutation led to a 10-fold increase in MalQ activity. These data suggest that the identified open reading frame encodes a repressor of the C. butyricum malQ gene, and of the adjacent malP gene. The gene has, therefore, been designated malR, and its encoded gene product MalR.     

Historical Contingency Causes Divergence in Adaptive Expression of the lac Operon

Populations of Escherichia coli selected in constant and fluctuating environments containing lactose often adapt by substituting mutations in the lacI repressor that cause constitutive expression of the lac operon. These mutations occur at a high rate and provide a significant benefit. Despite this, eight of 24 populations evolved for 8,000 generations in environments containing lactose contained no detectable repressor mutations. We report here on the basis of this observation. We find that, given relevant mutation rates, repressor mutations are expected to have fixed in all evolved populations if they had maintained the same fitness effect they confer when introduced to the ancestor. In fact, reconstruction experiments demonstrate that repressor mutations have become neutral or deleterious in those populations in which they were not detectable. Populations not fixing repressor mutations nevertheless reached the same fitness as those that did fix them, indicating that they followed an alternative evolutionary path that made redundant the potential benefit of the repressor mutation, but involved unique mutations of equivalent benefit. We identify a mutation occurring in the promoter region of the uspB gene as a candidate for influencing the selective choice between these paths. Our results detail an example of historical contingency leading to divergent evolutionary outcomes.     

Radiation-induced tetramer-to-dimer transition of Escherichia coli lactose repressor

The wild type lactose repressor of Escherichia coli is a tetrameric protein formed by two identical dimers. They are associated via a C-terminal 4-helix bundle (called tetramerization domain) whose stability is ensured by the interaction of leucine zipper motifs. Upon in vitro γ-irradiation the repressor losses its ability to bind the operator DNA sequence due to damage of its DNA-binding domains. Using an engineered dimeric repressor for comparison, we show here that irradiation induces also the change of repressor oligomerisation state from tetramer to dimer. The splitting of the tetramer into dimers can result from the oxidation of the leucine residues of the tetramerization domain.     

The possible roles of residues 79 and 80 of the Trp repressor from Escherichia coli K-12 in trp operator recognition

We constructed mutants of the Trp repressor from Escherichia coli K-12 with all possible single amino acid exchanges at positions 79 and 80 (residues 1 and 2 of the recognition helix). We tested these mutants in vivo by measuring the repression of synthesis of β-galactosidase with symmetric variants of α- and β-centered trp operators, which replace the lac operator in a synthetic lac system. The Trp repressor carrying a substitution of isoleucine 79 by lysine, showed a marked specificity change with respect to base pair 7 of the α-centered trp operator. Gel retardation experiments confirmed this result. Trp repressor mutant IR79 specifically recognizes a trp operator variant with substitutions in positions 7 and 8. Another mutant, with glycine in position 79, exhibited loss of contact at base pair 7. We speculate that the side chain of Ile79 interacts with the AT base pairs 7 and 8 of the α-centered trp operator, possibly with the methyl groups of thymines. Replacement of thymine in position 7 or 8 by uracil confirms the involvement of the methyl group of thymine 8 in repressor binding. Several Trp repressor mutants in position 80 (i.e. AI80, AL80, AM80 and AP80) broaden the specificity of the Trp repressor for α-centered trp operator variants with exchanges in positions 3, 4 and 5.