Lac repressor–operator interaction: N-terminal peptide backbone 1H and 15N chemical shifts upon complex formation with DNA

When the lac repressor tetramer is bound to its DNA operator, methylation protection shows the nearly symmetric operator half-sites are contacted asymmetrically. This asymmetric binding results from the DNA sequence/structure. The reported structure of lac repressor N-terminal fragment and an 11 base-pair operator left half-site provides no information concerning the effect of asymmetric binding, from left operator half-site to right half-site, upon the polypeptide backbone. We isolated uniformly ¹⁵N labeled 56 amino acid wild-type (HP56WT) and 64 residue mutant [Pro3>Tyr3] (HP64tyr3) lac repressor N-terminal DNA binding fragments for ¹H/¹⁵N NMR studies with the left and right operators separately. Spectral coincidence of these longer fragments, indicating structural similarity with a protease derived 51 amino acid fragment for which the amide correlations are assigned, allows for assignment of the common amide resonances. For both HP56WT and HP64tyr3, spectral overlap of the amide correlation peaks reveals the polypeptide backbones of the uncomplexed polypeptides are structurally similar. Likewise the complexes of the peptides to the 11 base-pair lac left operator half-site are similar. On the other hand, complexes of HP56WT and the left compared to the right lac operator half-site show different residues of the polypeptide are affected by binding different half-sites of the operator. Thus, the DNA sequence/structure transmits asymmetry to the polypeptide backbone of the interacting protein.     

Infrared study of the secondary structure of lac repressor and its tryptic core

The lac repressor and its tryptic core were studied by ir spectroscopy, and their β-structure content was determined by analysis of the spectra. Using protein-derived reference spectra, we find a β-content for lac repressor of 18% and of 23% for its tryptic core. The higher amount of β-conformation in the tryptic core is confirmed by another type of analysis (decomposition of the spectra in Gaussian curves). These results are discussed with respect to their implications for the structure of the N-terminal “headpiece” of lac repressor and for the mode of interaction of lac repressor with lac operator.     

NMR Investigation of Palindromic LAC Operator DNA Sequences of Varying Size: Influences of Complex Formation with the LAC Repressor Headpiece on the DNA 1H Resonances

Abstract

¹H-NMR studies on s3rmmetrical lac operators were performed to determine the minimum size of a lac operator for tight complex formation with the lac repressor headpiece. Four operators of varying size from 18 base pairs to 24 base pairs were chemically synthesized. The data obtained suggest that for a synthetic lac operator to form a tight complex with the lac headpiece it should be at least 22 base pairs long.     

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).     

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.     

Conformational prediction and circular dichroism studies on the lac repressor

Using the protein predictive model of Chou & Fasman (1974b), the secondary structure of the lac repressor has been elucidated from its amino acid sequence of 347 residues. The conformation is predicted to contain 37% α-helix and 35% β-sheet for the repressor, and 29% helix and 41% β-sheet for the trypsin-resistant core (residues 60 to 327). Circular dichroism studies indicate that native lac repressor contains 40% helix and 42% β-sheet, while the core has 16% helix and 54% β-sheet, in general agreement with the predicted conformation. The sharp reduction in helicity for the trypsinized lac repressor could be due to the loss of two long helical regions, 26–45 and 328–344, predicted at both terminals. There are extensive β-sheets predicted in the 215–324 region, which may be responsible for tetrameric stabilization found in both the lac repressor and the core. Residues 17 to 33 were previously predicted by Adler et al. (1972) to be helical and were proposed to bind in the major groove of DNA. However, the present analysis shows that there are two anti-parallel β-sheet regions: 4–7 and 17–24 at the N-terminal as well as 315–318 and 321–324 at the C-terminal of the lac repressor. These β-sheet pairs may assume the twisted “polypeptide double helix” conformation (Carter & Kraut, 1974) and bind to complementary regions in the major groove of DNA. The OH groups of Tyr at the N-terminal and those of Thr and Ser side chains, in both β-sheets at the N and C-terminal ends, could form hydrogen bonds to specific sites on the lac operator. There are 23 reverse β-turns predicted that may control the tertiary folding of the lac repressor, which is essential for operator binding. The behavior of several lac repressor mutants can be satisfactorily explained in terms of polar to non-polar group replacements as well as conformational changes in light of the present predicted model.     

Regulated autocrine growth of CHO cells

The goal of this work was to engineer a CHO cell line capable ofautocrine growth in a fully defined protein-free medium. Thiswas accomplished by stable integration of the genes encodinginsulin-like growth factor I (IGF-I) and transferrin into thegenome of a CHO-K1 cell line. Thelac operator/repressorsystem was used to regulate the expression of the IGF-I gene with thelac operator sequence being placed upstream ofthe coding sequence for IGF-I. The expression of thelacrepressor protein was driven by a modified metallothioneinpromoter allowing repressor expression to be regulated by theculture medium. The cell line calledSuper CHO^r (r for regulated) was able to grow in protein-free medium in an autocrine fashion with a doubling time of 20–24 hr,either attached to microcarriers or as aggregate suspensioncultures. Upon addition of metal to the culture medium, therepressor protein was produced and bound to the operatorsequences shutting down the expression of IGF-I and arrestingthe growth of the cells. Expression of the human growth hormone(hGH) gene and production of hGH was induced by the presence ofmetal ions. It was possible to release the cells from growtharrest in the presence of metal by the addition of isopropyl-D-thiogalactopyranoside (IPTG), which prevented bindingof the repressor to its operator sequences. The ability to growCHO cells in fully defined protein-free medium and to be able toregulate their growth rate offers a number of advantages for theuse of these cells as hosts for the production of recombinantDNA derived proteins.     

"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.     

1H, 13C and 15N backbone and side chain resonance assignments of a Myxococcus xanthus anti-repressor with no known sequence homologues

The CarS antirepressor activates a photo-inducible promoter in Myxococcus xanthus by physically interacting with the CarA repressor and eliminating the latter’s binding to operator DNA. Interestingly, interactions with both CarS and operator are crucially dependent on the DNA recognition helix of the CarA winged-helix DNA-binding domain. The CarA–CarS and the CarA-operator interfaces therefore overlap, and CarS may have structural features that mimic operator DNA. CarS has no known sequence homologues and its Gly and Pro contents are unusually high. Here, we report ¹H, ¹³C and ¹⁵N backbone and side chain assignments of CarS1, an 86-residue truncated yet fully functional variant of CarS. Secondary structural elements inferred from these data differ from those predicted from sequence.     

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 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.     

Repressor--operator interaction in the lac operon. II. Observations at the tyrosines and tryptophans

This paper shows that ¹⁹F-nuelear magnetic resonance spectroscopy on 3-fluoro-tyrosine and 5-fluorotryptophan-substituted wild-type lactose operon repressors from Escherichia coli can be used to examine the interactions with lac operator DNA.A survey of inducer and salt concentration effects on the repressor-operator complex is presented. The data lead us to a scheme for the interactions between the repressor, operator and inducer, in both binary and ternary complexes, that accommodate the results published by others.The complex between the tetrameric repressor and one 36 base-pair operator DNA fragment results in the simultaneous broadening of the resonances from all four N-terminal DNA binding domains. The actual contacts made by these binding domains are similar but probably not identical.The binding of the inducer molecule to the tetrameric repressor results in an allosteric change that can be monitored by the increased intensity of the resonances from individual tyrosine residues in the N-terminal binding domain. This increased N-terminal tyrosine resonance intensity in the complex is transmitted to repressor subunits that have not yet bound an inducer molecule.     

Mutational analysis of the operators of bacteriophage lambda

Summary O ^c mutations in the operators of bacteriophage lambda have been used to analyze the functional organization of the operators. In each operator, repressor binding sites 1 and 2, as identified biochemically, were found to be primarily responsible for the repressor affinity of the operators in vitro and for the repression of lytic functions in vivo. In addition, both sites were shown to be involved in the action of cro product at the operators. The data obtained have been used to estimate the repressor affinities of the individual binding sites. These affinities suggest that repressor bound at O _R1 and O _R2 interacts cooperatively. The results obtained support a model for repression of the early lambda operons where repressor bound at binding sites 1 and 2 interferes with RNA polymerase binding to the promoter sites.     

Prediction of 3-D Structure of the Cro Protein from Phage 434

Abstract

A comparative model building process has been utilized to predict (he three-dimensional structure of the bacteriophage 434 Cro protein, Amino acid sequence similarities between the 434 Cro protein and other bacteriophage repressor and Cro proteins have been used, in conjunction with secondary structure prediction and the known structures of other base sequence specific DNA binding proteins, to derive the model. From this model the interactions between the 434 Cro protein and its operator DNA have been deduced. These proposed interactions are consistent with the known properties of the bacteriophage 434 Cro protein.     

Neutron-scattering studies of lac repressor: A low-resolution model

A low-resolution model for the lac repressor is proposed from small-angle neutron studies on the native protein as well as on its isolated tryptic core and the 51 amino acids N-terminal peptide (headpiece). The implications for the interaction with the lac operator DNA are discussed.     

Two-Dimensional 1H, 15N NMR investigation of uniformly 15N-labeled Lac Repressor Headpiece

Abstract

¹⁵N uniformly labeled lac repressor and lac repressor headpiece were prepared. ¹⁵N NMR spectra of lac repressor were shown resolution inadequate for detailed study while the data showed that the ¹⁵N labeled N-terminal part of the protein is quite suitable for this type of study allowing future investigation of the specific interaction of the lac repressor headpiece with the lac operator. We report here the total assignment of proton ¹H and nitrogen ¹⁵NH backbone resonances of this headpiece in the free state. Assignments of the ¹⁵N resonances of the protein were obtained in a sequential manner using heteronuclear multiple quantum coherence (HMQC), relayed HMQC nuclear Overhauser and relayed HMQC-HOHAHA spectroscopy. More than 80 per cent of residues were assigned by their ¹⁵NH(i)-N¹H(i+1) and ¹⁵NH(i)-N¹(i-1) connectivities. Values of the ³J_NHα splitting for 39 of the 51 residues of the headpiece were extracted from HMQC and HMQC-J. The observed ¹⁵NH(i)-C^βH cross peaks and the ³J_NHα coupling constants values are in agreement with the three α-helices previously described [Zuiderweg, E.R.P., Scheek, R.M., Boelens, R., van Gunsteren, W.F. and Kaptein, R., Biochimie 67, 707 (1985)]. The ³J_NHα coupling constants can be now used for a more confident determination of the lac repressor headpiece. From these values it is shown that the geometry of the ends of the second and third α-helices exhibit deviation from the canonical α-helix structure. On the basis of NOEs and ³J_NHα values, the geometry of the turn of the helix-turn-helix motif is discussed.     

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.