Radiolysis of lac repressor by gamma-rays and heavy ions: a two-hit model for protein inactivation

Upon gamma-ray or argon ion irradiation of the lac repressor protein, its peptide chain is cleaved and the protein loses its lac operator-binding activity, as shown respectively by polyacrylamide gel electrophoresis and retardation gel electrophoresis. We developed phenomenological models that satisfactorily account for the experimental results: the peptide chain cleavage model considers that the average number of chain breaks per protomer is proportional to the irradiation dose and that the distribution of the number of breaks per protomer obeys Poisson's law. The repressor inactivation model takes into account the quaternary structure (a dimer of dimer) and the organization of the repressor in domains (two DNA binding sites, one per dimer). A protomer is inactivated by at least two different radiation-induced damages. A dimer is inactivated when at least one of the two protomers is inactivated. A tetramer is inactivated when both dimers are inactivated. From the combination of both models, we can deduce that chain cleavage cannot account for the protein inactivation, which should mainly result from oxidation of amino acid side chains. Indeed, particularly oxidizable and accessible amino acids (Tyr, His) are involved in the DNA binding process.     

Exposure of antigenic sites during immunization. Monoclonal antibodies to monomer of lactose repressor protein

Monoclonal antibodies specific for the lactose repressor protein have been purified from three mouse hybridoma cell lines, and ascitic fluids from five other cell lines producing repressor antibodies have been assayed for immunoglobulin subclass and antigenic specificity. The chymotryptic core region (amino acids 57-360) of the repressor reacted with all antibodies examined, while no reaction with the NH2-terminal domain (1-56) could be detected. All of the purified antibodies and ascitic fluids reacted with the carboxyl-terminal fragment (amino acids 281-360) produced by cyanylation and base-catalyzed cleavage at the cysteine residues. Although none of the purified antibodies associated with native, tetrameric lac repressor, reaction was observed with repressor which had been denatured or dissociated into monomers by treatment with low levels of sodium dodecyl sulfate. Additionally, a mutant repressor which exists as a monomer in solution reacted with the antibodies in the absence of any denaturing treatments. These data indicate the carboxyl-terminal region is inaccessible in the intact repressor tetramer and further suggest that denaturation/dissociation of a protein during the initial immunologic challenge may result in the production of monoclonal antibodies to antigenic areas of the protein which are not exposed in the native conformation.     

Recombination by resolvase is inhibited by lac repressor simultaneously binding operators between res sites

The Tn3 resolvase requires that the two recombination (res) sites be aligned as direct repeats on the same molecule for efficient recombination to occur. To test whether resolvase must contact the DNA between res sites as predicted by tracking models, we have determined the sensitivity of recombination to protein diffusion blockades. Recombination between two res sites is unaffected either by lac repressor or bacteriophage T7 RNA polymerase being bound between them. Yet recombination is inhibited by lac repressor if the res site is bounded by a lac operator on both sides. We demonstrate that lac repressor will bind to more than one DNA site under the conditions used to assay recombination. This result suggests that lac repressor can inhibit resolvase by forming a DNA loop that isolates a res site topologically. These results do not support a tracking model for resolvase but suggest that the structure and topology of the DNA substrate is important in the formation of a synapse between res sites.     

Construction, isolation and implications of repressor-galactosidase - beta-galactosidase hybrid molecules.

Escherichia coli heterogenotes, which produce hybrid molecules between the chimaeric protein repressor-galactosidase and the enzyme beta-galactosidase, were constructed. Repressor-galactosidase in which fully active lac repressor is covalently linked to active beta-galactosidase, is an aggregate with a core structure of four beta-galactosidase parts and two peripheral lac repressor dimers. The lac repressor dimers, which are separated by tetrameric beta-galactosidase, retain all the biological activities of tetrameric lac repressor. Substitution of repressor-galactosidase subunits with beta-galactosidase subunits leads to hybrid molecules with y beta-galactosidase subunits aggregated with (4-y) repressor-galactosidase subunits (where y = 1, 2 or 3). A 2:2 hybrid, i.e. a tetrameric beta-galactosidase core with one lac repressor dimer grafted to it, binds at least 100 times less strongly to 32P-labelled lambdaplac DNA than pure lac repressor or repressor-galactosidase. The data suggest a model in which lac repressor binds with two subunits to lac operator and with the other two subunits elsewhere on the DNA, possibly on sequences like the lac operator.     

Genetic studies of the lac repressor. XIII. Extensive amino acid replacements generated by the use of natural and synthetic nonsense suppressors

We have altered the amino acid sequence of the lac repressor one residue at a time by utilizing a collection of nonsense suppressors that permit the insertion of 13 different amino acids in response to the amber (UAG) codon, as well as an additional amino acid in response to the UGA codon. We used this collection to suppress nonsense mutations at 141 positions in the lacI gene, which encodes the 360 amino acid long lac repressor, including 53 new nonsense mutations which we constructed by oligonucleotide-directed mutagenesis. This method has generated over 1600 single amino acid substitutions in the lac repressor. We have cataloged the effects of these replacements and have interpreted the results with the objective of gaining a better understanding of lac repressor structure, and protein structure in general. The DNA binding domain of the repressor, involving the amino-terminal 59 amino acids, is extremely sensitive to substitution, with 70% of the replacements resulting in the I- phenotype. However, the remaining 301 amino acid core of the repressor is strikingly tolerant of substitutions, with only 30% of the amino acids introduced causing the I- phenotype. This analysis reveals the location of sites in the protein involved in inducer binding, tighter binding to operator and thermal stability, and permits a virtual genetic image reconstruction of the lac repressor protein.     

How Lac repressor finds lac operator in vitro.

Filter-binding and gel mobility shift assays were used to analyse the kinetics of the interaction of Lac repressor with lac operator. A comparison of the two techniques reveals that filter-binding assays with tetrameric Lac repressor have often been misinterpreted. It has been assumed that all complexes of Lac repressor and lac operator DNA bind with equal affinity to nitrocellulose filters. This assumption is wrong. Sandwich or loop complexes where two lac operators bind to one tetrameric Lac repressor are not or are only badly retained on nitrocellulose filters under normal conditions. Taking this into account, dimeric and tetrameric Lac repressor do not show any DNA-length dependence of their association and dissociation rate constants when they bind to DNA fragments smaller than 2455 base-pairs carrying a single symmetric ideal lac operator. A ninefold increased association rate to ideal lac operator on lambda DNA is observed for tetrameric but not dimeric Lac repressor. It is presumably due to intersegment transfer involving lac operator-like sequences.     

TET repressor.tet operator complex formation induces conformational changes in the tet operator DNA.

The structural changes of the tet operator DNA upon binding of the TET repressor protein are examined by circular dichroism. For this purpose a 70 bp DNA fragment was prepared which contains both tet operators. About 67% of the base pairs of this DNA are involved in specific interaction with the TET repressor. A rather large change in the CD of the DNA is induced by binding of the TET repressor. The shape of the CD difference spectrum is similar to the respective difference found for the lac operator DNA upon complex formation with the lac repressor. However, the effect induced by the TET repressor on tet operator DNA seems to comprise both the specific and non-specific effect of the lac repressor on the structure of DNA [Culard, F. and Maurizot, J.C. (1981) Nucl. Acids Res. 9, 5157-5184]. Specificity of binding is confirmed by the lack of any effect of the TET repressor on the CD of a 95 bp lac operator containing DNA fragment, by the reduced mobility of TET repressor.tet operator complexes on polyacrylamide gels under CD conditions, and by a titration experiment of tet operator DNA with TET repressor employing the CD change. The latter experiment reveals a stoichiometry of four TET repressors per tet operon control region.     

In vivo interaction of Escherichia coli lac repressor N-terminal fragments with the lac operator

Escherichia coli lac repressor is a tetrameric protein composed of 360 amino acid subunits. Considerable attention has focused on its N-terminal region which is isolated by cleavage with proteases yielding N-terminal fragments of 51 to 59 amino acid residues. Because these short peptide fragments bind operator DNA, they have been extensively examined in nuclear magnetic resonance structural studies. Longer N-terminal peptide fragments that bind DNA cannot be obtained enzymatically. To extend structural studies and simultaneously verify proper folding in vivo, the DNA sequence encoding longer N-terminal fragments were cloned into a vector system with the coliphage T7 RNA polymerase/promoter. In addition to the wild-type lacI gene sequence, single amino acid substitutions were generated at positions 3 (Pro3----Tyr) and 61 (Ser61----Leu) as well as the double substitution in a 64 amino acid N-terminal fragment. These mutations were chosen because they increase the DNA binding affinity of the intact lac repressor by a factor of 10(2) to 10(4). The expression of these lac repressor fragments in the cell was verified by radioimmunoassays. Both wild-type and mutant lac repressor N termini bound operator DNA as judged by reduced beta-galactosidase synthesis and methylation protection in vivo. These observations also resolve a contradiction in the literature as to the location of the operator-specific, inducer-dependent DNA binding domain.     

Activity changes in lac repressor with cysteine oxidation.

The effects of prior covalent cysteine modification or nonspecific DNA presence on the reaction of lac repressor protein with N-bromosuccinimide have been investigated. At low excesses, N-bromosuccinimide oxidation causes loss of operator DNA binding activity with simultaneous retention of inducer and nonspecific DNA binding activities. Cysteine and methionine are oxidized under the conditions utilized. Covalent modification of the cysteines of repressor prior to reaction decreased the observed loss of operator DNA binding capacity; the presence of nonspecific DNA partially prevented oxidation of the cysteines by N-bromosuccinimide, and concurrent protection of operator binding ability was observed. Methionine oxidation was observed in the cases where protection of the operator DNA binding capacity of repressor was seen. The region surrounding cysteine 107 was found to be influential in maintaining intact operator DNA binding function in repressor. This observation provides chemical evidence for the contribution of the core region of repressor in determining specificity of the protein in binding the lac operator. The protection from oxidation of cysteine residues in the core region by the presence of nonspecific DNA suggests that this binding influences the core region of the protein.     

Isolation of amino-terminal fragment of lactose repressor necessary for DNA binding.

lac repressor can be dissected by trypsin into a homogenous tetrameric core (accounting for residues 60 to 347), carrying inducer binding activity, and the monomeric amino-terminal peptides ("headpieces") accounting for residues 1 to 59 and 1 to 51, respectively. This restriction of the action of trypsin on lac repressor is obtained in 1 M Tris-HCl (pH 7.5)-30% in glycerol at 25 degrees C since only the peptide bonds at lysine-59 and to a lesser extent after at arginine-51 are cleaved under these conditions. The headpieces can be purified by gel filtration. They have ordered secondary structure as revealed by circular dichroism studies. The monomeric headpieces show the relatively weak binding to nonoperator DNA but not the highly specific and strong binding to operator DNA typical for tetrameric lac repressor.     

Lambda repressor inactivation: properties of purified ind- proteins in the autodigestion and RecA-mediated cleavage reactions

Under physiological conditions, lambda repressor can be inactivated in vivo or in vitro by RecA-mediated cleavage of the polypeptide chain. The repressor protein is thought to cleave itself, with RecA acting to stimulate autodigestion. ind- repressor mutants are resistant to RecA-mediated inactivation in vivo. In this paper, we report the purification of 15 ind- repressor proteins and the behaviors of these proteins in the RecA-mediated and autodigestion cleavage reactions. None of these proteins undergoes substantial RecA-dependent cleavage. However, eight mutant proteins autodigest at the same rate as wild-type repressor, six mutants do not autodigest or autodigest slower, and one mutant autodigests faster than wild-type. We discuss these results with respect to repressor structure and RecA-binding, and suggest possible roles for the RecA protein in the cleavage reaction.     

Stimulation of RecA-mediated cleavage of phage phi 80 cI repressor by deoxydinucleotides

The presence of either deoxyguanylyl-(3'----5')-deoxyguanosine (d(G-G] or deoxyadenylyl-(3'----5')-deoxyguanosine (d(A-G] greatly stimulates cleavage of the phage phi 80 cI repressor mediated by the Escherichia coli RecA protein in vitro. No other deoxydinucleoside monophosphate or riboguanylyl-(3'----5')-guanosine (r(G-G] affects the cleavage reaction. Neither the cleavage site of the phi 80 cI repressor nor the requirement for single-stranded DNA and ATP for cleavage is altered by d(G-G). Photoaffinity labeling experiments with 32P-labeled 5'-phosphoryl deoxyguanylyl deoxyguanosine (pd(G-G], which also stimulates cleavage, show that pd(G-G) bound to the repressor under the conditions in which the repressor is cleaved by RecA protein. The binding increases the affinity of the repressor for RecA protein and thus greatly stimulates repressor cleavage. The cleavage reactions of LexA and lambda cI repressors by RecA protein are not affected by d(G-G).     

Circular dichroism studies of the binding of o-nitrophenyl-beta-D-fucoside and o-nitrophenyl-beta-D-galactoside to lac repressor.

The binding of o-nitrophenyl-beta-D-fucoside and o-nitrophenyl-beta-D-galactoside to Escherichia coli lac repressor was investigated by circular dichroism in the wavelength range 300--400 nm corresponding to the o-nitrophenyl chromophores. The CD signal of both ligands drastically changed when they bound to lac repressor due to the asymmetric interaction of the o-nitrophenyl ring with chemical groups of protein. The CD spectra of bound ligands indicate close similarity between the environment of o-nitrophenyl-beta-D-fucoside and o-nitrophenyl-beta-D-galactoside on lac repressor. The CD signal is used to calculate the binding parameters (K and n) to lac repressor. It is demonstrated that the limited proteolytic digestion of lac repressor which gives a 'core protein' does not affect the environment of both ligands on the protein.     

Synthetic lac operator mediates repression through lac repressor when introduced upstream and downstream from lac promoter.

Plasmids were constructed which carry a synthetic lac operator at various distances from the lac promoter. They were tested in vivo for function in the presence and absence of lac repressor. We found significant repression when the lac operator is situated at the 3' end of the lac I gene or at the 5' end of the lac Z gene. When lac operators are inserted at both sites, we found a greater than 150-fold repression. The complex between lac repressor and DNA carrying these two lac operators is exceedingly stable in vitro suggesting that one tetrameric lac repressor may bind to both lac operators.     

Achilles' heel cleavage: creation of rare restriction sites in lambda phage genomes and evaluation of additional operators, repressors and restriction/modification systems

A novel technique for the creation of rare restriction sites was described by Koob et al. [Science 241 (1988) 1084-1086]. This technique, Achilles' heel cleavage (AC), relies on the use of a bound repressor molecule to protect only one of many identical restriction sites from a modification methyltransferase that inactivates all other restriction sites. The technique was applied to a small plasmid and shown to work efficiently with two repressor/operator systems: lac repressor/lacO operator and lambda repressor/lambda oL1 operator. Here, we have extended these results to a lac operator carried by a much larger vector, namely a 44-kb phage lambda construct. In addition, we have evaluated the effect of altering the stability of the lac repressor/lac operator complex by varying both the operator and the repressor. We have also evaluated several more restriction/modification systems (MboI, Dam, MspI and AluI) in addition to HhaI and HaeII used earlier. Finally, we extended the AC technique to a third system, that of the phage 434 repressor and a synthetic 434 operator. From our results we conclude that the AC method should be applicable to the mapping of large genomes and to measuring the strength of operator-repressor interactions. AC could also be applied to identifying and evaluating many different DNA-binding proteins and their sites of action.     

Interaction of effecting ligands with lac repressor and repressor-operator complex.

The equilibrium association constants for the binding of a wide variety of effecting ligands of the lac repressor were measured by equilibrium dialysis. Also, detailed investigations of the apparent rate of dissociation of repressor-operator comples as a function of ligand concentration were carried out for several inducers and anti-inducers. The affinity of repressor-ligand comples for operator DNA was evaluated from the specific rate constants at saturating concentrations of effecting ligand. By fitting the experimental data depicting the functional dependence of the rate of dissociation upon ligand concentrations to calculated curves, assuming simple models of the induction mechanism, the equilibrium association constant for the binding of effecting ligand to repressor-operator comples was determined. Inducers reduce the affinity of lac repressor for operator DNA by a factor of approximately 1000 under standard conditions; the extent of destabilization depends on Mg2+ ion concentration. Anti-inducers increase the affinity of repressor for operator at most a factor of five. Only one neutral ligand, which binds to repressor without altering the stability of repressor-operator comples, was found. No homotropic or heterotropic interactions in the binding of effecting ligands either to repressor or to repressor-operator complex are evident.     

The interaction of the recognition helix of lac repressor with lac operator.

We have constructed a system which allows systematic testing of repressor--operator interactions. The system consists of two plasmids. One of them carries a lac operon in which lac operator has been replaced by a unique restriction site into which synthetic operators can be cloned. The other plasmid carries the gene coding for the repressor, in our case a semisynthetic lacI gene of which parts can be exchanged in a cassette-like manner. A galE host allows us to select for mutants which express repressors with altered specificities. Here we report the change of specificity in the lac system by changing residues 1 and 2 of the recognition helix of lac repressor. The specificity changes are brought about cooperatively by the change of both residues. Exchanges of just one residue broaden the specificity. Our results hint that the recognition helix of lac repressor may possibly have the opposite orientation to those in Lambda cro protein or 434 CI repressor.