Specificity and Affinity of Lac Repressor for the Auxiliary Operators O2 and O3 Are Explained by the Structures of Their Protein-DNA Complexes

The structures of a dimeric mutant of the Lac repressor DNA-binding domain complexed with the auxiliary operators O2 and O3 have been determined using NMR spectroscopy and compared to the structures of the previously determined Lac-O1 and Lac-nonoperator complexes. Structural analysis of the Lac-O1 and Lac-O2 complexes shows highly similar structures with very similar numbers of specific and nonspecific contacts, in agreement with similar affinities for these two operators. The left monomer of the Lac repressor in the Lac-O3 complex retains most of these specific contacts. However, in the right half-site of the O3 operator, there is a significant loss of protein-DNA contacts, explaining the low affinity of the Lac repressor for the O3 operator. The binding mode in the right half-site resembles that of the nonspecific complex. In contrast to the Lac-nonoperator DNA complex where no hinge helices are formed, the stability of the hinge helices in the weak Lac-O3 complex is the same as in the Lac-O1 and Lac-O2 complexes, as judged from the results of hydrogen/deuterium experiments.     

Plasticity in protein-DNA recognition: lac repressor interacts with its natural operator 01 through alternative conformations of its DNA-binding domain

The lac repressor-operator system is a model system for understanding protein-DNA interactions and allosteric mechanisms in gene regulation. Despite the wealth of biochemical data provided by extensive mutations of both repressor and operator, the specific recognition mechanism of the natural lac operators by lac repressor has remained elusive. Here we present the first high-resolution structure of a dimer of the DNA-binding domain of lac repressor bound to its natural operator 01. The global positioning of the dimer on the operator is dramatically asymmetric, which results in a different pattern of specific contacts between the two sites. Specific recognition is accomplished by a combination of elongation and twist by 48 degrees of the right lac subunit relative to the left one, significant rearrangement of many side chains as well as sequence-dependent deformability of the DNA. The set of recognition mechanisms involved in the lac repressor-operator system is unique among other protein-DNA complexes and presents a nice example of the adaptability that both proteins and DNA exhibit in the context of their mutual interaction.     

Novel method for identifying sequence-specific DNA-binding proteins.

We developed a general method for the enrichment and identification of sequence-specific DNA-binding proteins. A well-characterized protein-DNA interaction is used to isolate from crude cellular extracts or fractions thereof proteins which bind to specific DNA sequences; the method is based solely on this binding property of the proteins. The DNA sequence of interest, cloned adjacent to the lac operator DNA segment is incubated with a lac repressor-beta-galactosidase fusion protein which retains full operator and inducer binding properties. The DNA fragment bound to the lac repressor-beta-galactosidase fusion protein is precipitated by the addition of affinity-purified anti-beta-galactosidase immobilized on beads. This forms an affinity matrix for any proteins which might interact specifically with the DNA sequence cloned adjacent to the lac operator. When incubated with cellular extracts in the presence of excess competitor DNA, any protein(s) which specifically binds to the cloned DNA sequence of interest can be cleanly precipitated. When isopropyl-beta-D-thiogalactopyranoside is added, the lac repressor releases the bound DNA, and thus the protein-DNA complex consisting of the specific restriction fragment and any specific binding protein(s) is released, permitting the identification of the protein by standard biochemical techniques. We demonstrate the utility of this method with the lambda repressor, another well-characterized DNA-binding protein, as a model. In addition, with crude preparations of the yeast mitochondrial RNA polymerase, we identified a 70,000-molecular-weight peptide which binds specifically to the promoter region of the yeast mitochondrial 14S rRNA gene.     

Role of the purine repressor hinge sequence in repressor function.

A protease-hypersensitive hinge sequence in Escherichia coli purine repressor (PurR) connects an N-terminal DNA-binding domain with a contiguous corepressor-binding domain. Binding of one molecule of dimeric repressor to operator DNA protects the hinge against proteolytic cleavage. Mutations in the hinge region impair repressor function in vivo. Several nonfunctional hinge mutants were defective in low-affinity binding to operator DNA in the absence of corepressor as well as in high-affinity corepressor-dependent binding to operator DNA, although binding of corepressor was similar to binding of the wild-type repressor. These results establish a role for the hinge region in operator binding and lead to a proposal for two routes to form the holoPurR-operator complex.     

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.     

A closer view of the conformation of the Lac repressor bound to operator

Crystal structures of the Lac repressor, with and without isopropyithiogalactoside (IPTG), and the repressor bound to operator have provided a model for how the binding of the inducer reduces the affinity of the repressor for the operator. However, because of the low resolution of the operator-bound structure (4.8 A), the model for the allosteric transition was presented in terms of structural elements rather than in terms of side chain interactions. Here we have constructed a dimeric Lac repressor and determined its structure at 2.6 A resolution in complex with a symmetric operator and the anti-inducer orthonitrophenylfucoside (ONPF). The structure enables the induced (IPTG-bound) and repressed (operator-bound) conformations of the repressor to be compared in atomic detail. An extensive network of interactions between the DNA-binding and core domains of the repressor suggests a possible mechanism for the allosteric transition.     

The lac repressor hinge helix in context: The effect of the DNA binding domain and symmetry

The Lac system of genes has been an important model system in understanding gene regulation. When the dimer lac repressor protein binds to the correct DNA sequence, the hinge region of the protein goes through a disorder to order transition. The hinge region is disordered when binding to nonoperator sequences. This region of the protein must pay a conformational entropic penalty to order when it is bound to operator DNA. Structural studies show that this region is flexible. Previous simulations showed that this region is disordered when free in solution without the DNA binding domain present. Our simulations corroborate that this region is extremely flexible in solution, but we find that the presence of the DNA binding domain proximal to the hinge helix and salt make the ordered conformation more favorable even without DNA present.     

The detection of DNA-binding proteins by protein blotting.

A method, called "protein blotting," for the detection of DNA-binding proteins is described. Proteins are separated on an SDA-polyacrylamide gel. The gel is sandwiched between 2 nitrocellulose filters and the proteins allowed to diffuse out of the gel and onto the filters. The proteins are tightly bound to each filter, producing a replica of the original gel pattern. The replica is used to detect DNA-binding proteins, RNA-binding proteins or histone-binding proteins by incubation of the filter with [32P]DNA, [125I]RNA, or [125I] histone. Evidence is also presented that specific protein-DNA interactions may be detected by this technique; under appropriate conditions, the lac repressor binds only to DNA containing the lac operator. Strategies for the detection of specific protein-DNA interactions are discussed.     

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.     

The role of the minor groove substituents in indirect readout of DNA sequence by 434 repressor

The sequence of non-contacted bases at the center of the 434 repressor binding site affects the strength of the repressor-DNA complex by influencing the structure and flexibility of DNA (Koudelka, G. B., and Carlson, P. (1992) Nature 355, 89-91). We synthesized 434 repressor binding sites that differ in their central sequence base composition to test the importance of minor groove substituents and/or the number of base pair hydrogen bonds between these base pairs on DNA structure and strength of the repressor-DNA complex. We show here that the number of base pair H-bonds between the central bases apparently has no role in determining the relative affinity of a DNA site for repressor. Instead we find that the affinity of DNA for repressor depends on the absence or presence the N2-NH(2) group on the purine bases at the binding site center. The N2-NH(2) group on bases at the center of the 434 binding site appears to destabilize 434 repressor-DNA complexes by decreasing the intimacy of the specific repressor-DNA contacts, while increasing the reliance on protein contacts to the DNA phosphate backbone. Thus, the presence of an N2-NH(2) group on the purines at the center of a binding site globally alters the precise conformation of the protein-DNA interface.     

Determinants of bacteriophage 933W repressor DNA binding specificity

We reported previously that 933W repressor apparently does not cooperatively bind to adjacent sites on DNA and that the relative affinities of 933W repressor for its operators differ significantly from that of any other lambdoid bacteriophage. These findings indicate that the operational details of the lysis-lysogeny switch of bacteriophage 933W are unique among lambdoid bacteriophages. Since the functioning of the lysis-lysogeny switch in 933W bacteriophage uniquely and solely depends on the order of preference of 933W repressor for its operators, we examined the details of how 933W repressor recognizes its DNA sites. To identify the specificity determinants, we first created a molecular model of the 933W repressor-DNA complex and tested the predicted protein-DNA interactions. These results of these studies provide a picture of how 933W repressor recognizes its DNA sites. We also show that, opposite of what is normally observed for lambdoid phages, 933W operator sequences have evolved in such a way that the presence of the most commonly found base sequences at particular operator positions serves to decrease, rather than increase, the affinity of the protein for the site. This finding cautions against assuming that a consensus sequence derived from sequence analysis defines the optimal, highest affinity DNA binding site for a protein.     

The Lac repressor: a second generation of structural and functional studies

In the past year, the crystal structure of a dimeric version of the Escherichia coli Lac repressor bound to operator DNA was determined at 2.6A resolution, providing a closer view of the operator-bound conformation of the repressor. Refined NMR studies of the DNA-binding portion of the repressor complexed to operator DNA have revealed further details of the unique DNA-binding interactions of the repressor. The structural studies have been complemented by continued biochemical studies, with the overall goal of understanding the mechanism of allosteric regulation.     

Determination of the molecular weight of DNA-bound protein(s) responsible for gel electrophoretic mobility shift of linear DNA fragments examplified with purified viral myb protein.

A protein-DNA complex has less gel electrophoretic mobility than the free DNA fragment. One parameter for the degree of retardation of a linear DNA fragment in a protein-DNA complex is the molecular weight of the bound protein(s). The quotient of the migration distances of free DNA (m) and protein-DNA complex (m') is a function of the molecular weight (MW) of the bound protein(s). Based on the evaluation of the lac repressor induced mobility shift of a 203 bp DNA fragment containing the lac operator in a 5% non-denaturating polyacrylamide gel a direct proportionality could be shown between (m/m'-1) and MW with the proportionality factor K = 215 kDa. The factor K depends on the acrylamide concentration in the gel, getting lower values with increasing acrylamide concentrations. A calculation is given to determine the molecular weight of DNA-binding factors responsible for the decreased electrophoretic mobility of a linear DNA fragment. As an example this calculation was used in order to analyse DNA-binding of the isolated viral myb protein. It could be demonstrated that the viral myb protein binds to DNA as a monomer and as a dimer.     

Orientation of the putative recognition helix in the DNA-binding domain of Hin recombinase complexed with the hix site

On the basis of sequence similarity with other known DNA-binding proteins, the DNA-binding domain of Hin recombinase, residues 139-190, is thought to bind DNA by a helix-turn-helix motif. Two models can be considered that differ in the orientation of the recognition helix in the major groove of DNA. One is based on the orientation of the recognition helix found in the 434 repressor (1-69) and lambda repressor-DNA cocrystals, and the other is based on the NMR studies of lac repressor headpiece. Cleavage by EDTA.Fe attached to a lysine side chain (Ser183----Lys183) near the COOH terminus of Hin(139-184) reveals that the putative recognition helix is oriented toward the center of the inverted repeats in a manner similar to that seen in the 434 and lambda repressor-DNA cocrystals.     

Crystallographic analysis of Lac repressor bound to natural operator O1

Previous structures of Lac repressor bound to DNA used a fully symmetric "ideal" operator sequence that is missing the central G-C base-pair present in the three natural operator sequences. Here we have determined the X-ray crystal structure of a dimeric Lac repressor bound to a 22 base-pair DNA with the natural operator O1 sequence and the anti-inducer ONPF, at 4.0 A resolution. The natural operator is bent in the same way as the symmetric sequence, due to the binding of the hinge helices of the repressor to the minor groove at the central GCGG sequence of O1. Comparison of the structures of the repressor bound to the natural and symmetric operators shows very similar overall structures, with only slight rearrangements of the headpiece domains of the repressor. Analysis of crystals with iodinated DNA shows that the operator is uniquely positioned and allows for the sequence registration of the DNA relative to the repressor to be determined. The kink in the operator is centered between the left half-site and the central G-C base-pair of O1. Our results are most consistent with a previously proposed model in which, relative to the complex with the symmetric operator, the repressor accommodates binding to the natural operator sequence by shifting the position of the right headpiece by one base-pair step towards the center of O1.     

In vivo HP1 targeting causes large-scale chromatin condensation and enhanced histone lysine methylation

Changes in chromatin structure are a key aspect in the epigenetic regulation of gene expression. We have used a lac operator array system to visualize by light microscopy the effect of heterochromatin protein 1 (HP1) alpha (HP1alpha) and HP1beta on large-scale chromatin structure in living mammalian cells. The structure of HP1, containing a chromodomain, a chromoshadow domain, and a hinge domain, allows it to bind to a variety of proteins. In vivo targeting of an enhanced green fluorescent protein-tagged HP1-lac repressor fusion to a lac operator-containing, gene-amplified chromosome region causes local condensation of the higher-order chromatin structure, recruitment of the histone methyltransferase SETDB1, and enhanced trimethylation of histone H3 lysine 9. Polycomb group proteins of both the HPC/HPH and the EED/EZH2 complexes, which are involved in the heritable repression of gene activity, are not recruited to the amplified chromosome region by HP1alpha and HP1beta in vivo targeting. HP1alpha targeting causes the recruitment of endogenous HP1beta to the chromatin region and vice versa, indicating a direct interaction between the two HP1 homologous proteins. Our findings indicate that HP1alpha and HP1beta targeting is sufficient to induce heterochromatin formation.