Binding of lac repressor to the secondary lac operator in Escherichia coli.

Summary In the lac operon, the existence of a secondary repressor binding site, inside Z gene, had been inferred from in vitro binding studies (Reznikoff et al., 1974; Gilbert et al., 1975).A serie of deletions have been constructed from a lac transducing bacteriophage. Some of those deleted bacteriophages have still the property of derepressing a chromosomal lac operon, even though they do not contain any more the lac operator. This phenomenon is an indication that the secondary repressor binding site is also active in vivo.     

Studies on gene control regions XII. The functional significance of a lac operator constitutive mutation.

The functional significance of a lac operator constitutive mutation has been determined. The transition adenine-thymine to guanine-cytosine was shown to be a constitutive mutation simply because thymine contains the functionally important 5-methyl group whereas cytosine does not. The remainder of the base pair is of no consequence. The experimental approach was to synthesize various modified operators containing cytosine, 5-methyl-cytosine, and 5-bromocytosine. The synthetic operator containing a guanine-cytosine base pair displays an eightfold reduction in stability with lac repressor whereas the operator containing 5-methylcytosine binds repressor at least as tightly as does the wild type sequence. Results published previously have shown that a similar decrease in stability of the repressor-operator complex can be obtained simply by substituting uracil for thymine or by inverting the base pair to thymine-adenine. All these results taken together implicate the thymine 5-methyl as the only important functional group recognized by the lac repressor at this base pair. Further confirmation of this conclusion was obtained by substitution of 5-bromocytosine and 5-bromouracil at this base pair. Both altered the stability of the repressor-operator complex by about the same percent suggesting that the bromine atom was the important determinant of complex stability for 5-bromopyrimidine analogs.     

Studies on gene control regions. 1. Chemical synthesis of lactose operator deoxyribonucleic acid segments.

The chemical synthesis of lactose operator DNA segments is described. The 31-base-paired duplex contains the DNA recognized by lac repressor protein and twofold rotationally symmetric base pairs on either side of the tight binding region. The synthesis includes the deoxyoligonucleotides d(T-G-T-G-G), d(A-A-T-T-G-T-G-A-G), d(C-G-G-A-T-A-A-C-A-A-T-T), d(T-C-A-C-A), d(T-G-T-G-A-A-A-T-T-G-T), d(T-A-T-C-C-G-C-T-C-A-C), and d(A-A-T-T-C-C-A-C-A). These deoxyoligonucleotides were characterized by two-dimensional sequencing techniques, paper chromatography, and thin-layer chromatography.     

Studied on gene control regions IX. The effect of hypoxanthine-substituted lac operators on the lac operator--lac repressor interaction.

Hypoxanthine was substituted for guanine at specific sites in the lac operator DNA by a combination of chemical and enzymatic procedures. The stability of these modified lac operators with wild-type (SQ) and tight binding (QX86) lac repressors was measured. Effects were variable. At some sites insertion of hypoxanthine significantly reduced the stability of the complex whereas at other sites substitution with hypoxanthine did not alter the repressor—operator interaction. In addition, insertion of this analog at two sites increased the stability of the complexes. These changes were used to partially map regions of the lac operator that are in contact with lac represser. The results suggest that lac repressor recognizes the guanine 2-amino group at specific sites in the minor groove of lac operator.     

Studies on gene control regions. VI. The 5- methyl of thymine, a lac repressor recognition site.

Three site specific deoxyuridine analogs of lac operator were tested for binding with wild type (SQ) and tight binding (QX86) lac repressors. Insertion of uracil for thymine at site 13 (our nomenclature) significantly reduced the dissociation half-life of QX86 repressor for lac operator DNA (21 vs 1.2 min). Two other sites (6 and 7) are affected to a much lesser extent.     

Studies on gene control regions X. The effect of specific adenine-thymine transversions on the lac repressor-lac operator interaction.

Chemical and enzymatic methods were used to synthesize a transition (AT to GC) and a transversion (AT to TA) at a lac operator site known to interact with lac repressor through the thymine 5 methyl group. These operators also contained a poly(dA) . poly(dT) tail 8 to 12 base pairs in length at one end. Results suggest that the steric constraints of lac repressor relative to the position of the 5 methyl group are quite critical. For example a seven fold reduction in stability was observed for the transversion. Results also suggest that the operator spans at least 21 base pairs.     

A model for the binding of lac repressor to the lac operator

A model is suggested for the lac repressor binding to the lac operator in which the repressor polypeptide chain sequences from Gly 14 to Ala 32 and from Ala 53 to Leu 71 are involved in specific interaction with operator DNA. A correspondence between the protein and DNA sequences is found which explains specificity of the repressor binding to the lac operator. The model can be extended to describe specific binding of other regulatory proteins to DNA.     

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.     

DNA-binding site of lac repressor probed by dimethylsulfate methylation of lac operator.

In order to compare the structures of the DNA-binding sites on variants of the lac repressor, we have studied the influence of these variants on the dimethylsulfate methylation of the lac operator. Since a bound protein changes the availability of specific purines in the operator to this chemical attack, comparisons of the methylation patterns will show similarities or differences in the protein DNA contacts. We compared lac repressor, induced lac repressor (repressor bound to the gratuitous inducer isopropyl-β-d-thiogalactoside), mutant repressors having increased operator affinities (X86, I12 and the X86-I12 double mutant) and repressor peptides (long headpiece, residues 1 to 59 and short headpiece. residues 1 to 51). All of these repressors and repressor peptides exhibit the same general pattern of protection and enhancement in the operator; however, the short headpiece pattern differs most from that of the repressor while the induced repressor and the long headpiece show intermediate patterns that are strikingly similar to each other. The mutant repressors do not show an isopropyl-β-d-thiogalactoside effect but otherwise are almost indistinguishable from wild-type repressor. These results demonstrate that all molecules bind to the operator using basically the same protein-DNA contacts; they imply that (1) most and possibly all repressor contacts to operator lie within amino acids 1 to 51, (2) inducer weakens many contacts rather than totally disrupting one or even a few and (3) the tight-binding mutants do not make additional contacts to the DNA.These results are consistent with a model in which the amino-terminal portions of two repressor monomers make the DNA contacts. We show that one can understand the affinity of binding as related to the accuracy of the register of the two amino-terminal portions along the DNA. Furthermore, the action of inducer and the behaviour of the tight binding mutants can be accomodated within a two-state model in which the strongly or weakly binding states correspond to structures in which the amino-terminal regions are rigidly or loosely held with respect to each other.     

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   

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.     

A comparison of lac repressor binding to operator and to nonoperator DNA

We have compared the operator and nonoperator DNA binding activities of the lac repressor with respect to inactivation or inhibition by trypsin, heat, actinomycin, and isopropylthiogal-actoside. The two DNA binding activities were found to differ only in their sensitivity to the inducing ligand isopropylthiogal-actoside. Repressor binding to poly(dT-dT-dG)·poly(dC-dA-dA) was shown not to be affected by isopropylthiogalactoside.     

NMR study of the interaction between the lac repressor and the lac operator

Binding of the lac repressor headpiece, the N-terminal region of the lac repressor, to the lac operator of Escherichia coli was studied by 1H-NMR spectroscopy. Two DNA fragments, of 51 base pairs and 62 base pairs, containing the lac operator region, were investigated. The signals of their hydrogen-bonded imino protons were well resolved in the 500-MHz NMR spectra. The spectra of the free lac operator DNA are similar to those obtained from ring-current-shift calculations for a B-DNA structure. Complex formation with the headpiece led to small but nevertheless characteristic changes in the spectra. The fact that very few imino resonances shifted upon addition of headpiece, as well as the variety in direction and size of these chemical shifts, indicate the formation of a specific complex between the lac repressor and the lac operator. The observed changes in the resonance positions exclude the intercalation of tyrosine residues of the headpiece between adjacent base pairs of the lac operator as well as the formation of a cruciform structure. They rather reflect a small conformational transition in the DNA itself, caused for example by an alteration in the tilt of a few base pairs or a shift of the keto-enol tautomeric equilibrium of the bases towards the enolic form.     

Photo-CIDNP study of the interaction between lac repressor headpiece and lac operator DNA

Lac repressor headpiece (HP) and intact lac repressor have been studied using the photo-CIDNP method. At neutral pH histidine 29, tyrosines 7, 12 and 17 and methionine 1 are polarised. His-29 polarizations are weaker and broader in HP59 than in HP51 indicating that the C-terminal octapeptide in HP59 adopts a conformation that allows an interaction with His-29. The photo-CIDNP spectra of intact lac repressor and HP51 are very similar, showing that the same residues are accessible to the photo-excited flavin. An equimolar mixture of HP51 and a 14 base pair lac operator fragment strongly suppresses the photo-CIDNP effect of tyrosines 7 and 17 and abolishes the His-29 polarizations. The results are compared with earlier photo-CIDNP measurements on a complex of headpiece with poly[d(AT)] and with a model derived from a 2D NMR study on a lac headpiece-operator complex.     

Tryptic core protein of lactose repressor binds operator DNA.

The core protein produced by mild proteolytic digestion of lactose repressor protein has been purified from native repressor by chromatography on phosphocellulose. The core protein isolated in this manner binds to operator DNA with an apparent dissociation constant of 10(-7) M, and the observed binding is decreased by the presence of inducer. Competition studies with nonspecific DNA indicate that the binding species in the core protein preparations is neither intact lactose repressor nor mixed tetramers containing varying numbers of intact NH2-terminal regions. This conclusion is supported by experiments designed to measure the rate of dissociation of the core protein from the operator DNA. Calculations based on the assumption that the isolated core protein binds similarly to the corresponding region in intact repressor protein indicate that the core region contributes approximately 40 to 50% of the energy of binding to operator DNA. Furthermore, the change in operator affinity upon inducer binding to core accounts for a minimum of 60% of the free energy change in binding to operator observed for the native protein. The demonstration that core protein binds to operator DNA requires a re-evaluation of the various models for repressor binding to DNA. A possible model based on the available information is presented.     

Allosteric regulation of inducer and operator binding to the lactose repressor

The effects of cysteine modification and variations in pH on the equilibrium parameters for inducer and operator binding to the lactose repressor protein were examined. Operator binding affinity was minimally affected by increasing the pH from 7.5 to 9.2, whereas inducer binding was decreased for both the unliganded protein and the repressor-operator complex over the same range. Inducer binding to the repressor became more cooperative at high pH. The midpoint for the change in inducer affinity and cooperativity was pH 8.3; this value correlates well with cysteine ionization. The differential between repressor-operator affinity in the presence and absence of inducer was significantly decreased by modification of the protein with methyl methanethiosulfonate (MMTS). In contrast to unreacted protein, the inducer binding parameters for MMTS-modified repressor were largely unaffected by pH variation. The free energy for formation of the completely liganded protein was calculated for two pathways; the delta G values for these two independent routes were equivalent only for stoichiometries of four inducers and two operators per repressor molecule. All of the binding data were analyzed quantitatively by using a Monod-Wyman-Changeux two-state model for allosteric regulation. The observed dependences of the isopropyl beta-D-thiogalactoside binding curves on pH, DNA concentration, and MMTS modification were fitted by varying only the equilibrium constant between the two conformational states of the protein. With this analysis, high pH favors the T (high operator/low inducer affinity) state, while modification of cysteine-281 with MMTS elicits a shift into the R (high inducer/low operator affinity) state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lac repressor with the helix-turn-helix motif of lambda cro binds to lac operator.

Lac repressor, lambda cro protein and their operator complexes are structurally, biochemically and genetically well analysed. Both proteins contain a helix-turn-helix (HTH) motif which they use to bind specifically to their operators. The DNA sequences 5'-GTGA-3' and 5'-TCAC-3' recognized in palindromic lac operator are the same as in lambda operator but their order is inverted form head to head to tail to tail. Different modes of aggregation of the monomers of the two proteins determine the different arrangements of the HTH motifs. Here we show that the HTH motif of lambda cro protein can replace the HTH motif of Lac repressor without changing its specificity. Such hybrid Lac repressor is unstable. It binds in vitro more weakly than Lac repressor but with the same specificity to ideal lac operator. It does not bind to consensus lambda operator.