Imino proton assignments in the proton nuclear magnetic resonance spectrum of the lambda phage OR3 deoxyribonucleic acid fragment

The 17 base pair duplex d(TATCACCGCAAGGGATAp) . d(TATCCCTTGCGGTGATAp) corresponding to the OR3 operator site of lambda phage has been synthesized and studied by 1H nuclear magnetic resonance spectroscopy at 470 MHz. The 13 imino proton resonances observed at 20 degrees C have been assigned to specific base pairs at positions 3-15 on the basis of nuclear Overhauser effect measurements and studies of the temperature dependence of peak intensities. Resonances from the A-T base pairs at positions 1, 2, 16, and 17 are assumed to be absent from the spectrum because of terminal fraying. Resonance from many of the base pairs suggested by Ohlendorf et al. [Ohlendorf, D. H., Anderson, W. F., Fisher, R. G., Takeda, Y., & Matthews, B. W. (1982) Nature (London) 298, 718-723] to be involved in specific binding of the lambda phage cro repressor are well resolved.     

Lambda cro repressor complex with OR3 DNA: 15N NMR observations

15N NMR studies of the coliphage lambda cro repressor are presented. The protein has been uniformally labeled with 15N, and individual amino acids have been incorporated. Although the four C-terminal residues (63-66) were not located in the original crystallographic studies of the protein [Anderson, W.F., Ohlendorf, D.H., Takeda, Y., & Matthews, B.W. (1981) Nature (London) 290, 754], it has been proposed that the C-terminus is involved in DNA binding [Ohlendorf, D.H., Anderson, W.F., Fisher, R.G., Takeda, Y., & Matthews, B.W. (1982) Nature (London) 298, 718]. These experiments give direct verification of that proposal. [15N]Amide resonances are assigned for residues 56, 62, 63, and 66 in the C-terminus by enzymatic digestion and by 13C-15N double-labeling experiments. 15N[1H] nuclear Overhauser effects show that the C-terminus is mobile on a nanosecond time scale. Exchange experiments using distortionless enhancement via polarization transfer, which is sensitive to proton exchange on the 1/JNH (10 ms) time scale, indicate that the amide protons in the C-terminus are freely accessible to solvent. It is thus a flexible arm in solution. The binding of both specific operator and nonspecific DNA is shown to reduce both the mobility and the degree of solvent exposure of this arm. Two-dimensional 15N-1H correlation experiments using 15N-labeled cro reveal inconsistencies with previously reported 1H NMR assignments for the lysine amides [Weber, P.L., Wemmer, D.E., & Reid, B.R. (1985) Biochemistry 24, 4553]. This result suggests that those assignments require reexamination, illustrating the utility of 15N labeling for obtaining 1H resonance assignments of biomolecules. Furthermore, isomerization of the peptide bond of Pro-59, which has been previously suggested (Weber et al., 1985) and which would significantly affect the properties of the C-terminal arm, is shown to not occur.     

Different interactions used by Cro repressor in specific and nonspecific DNA binding

The mode of interaction of Cro repressor with specific and nonspecific sites on DNA was explored by chemical modification and protection of lysine and tyrosine residues. Cro has 8 lysines. In the presence of DNA, lysines 32 and 56 are fully protected and lysines 21, 62, and 63 are partially protected from alkylation. However, the terminal amino group and lysines 8, 18, and 39 are not protected. Location of the protected and unprotected lysines on the three-dimensional Cro structure defines a DNA-binding region. The results provide direct experimental support for a mode of interaction between Cro and DNA, in which Cro buries its 2-fold related alpha-helices in consecutive DNA major grooves (Anderson, W. F., Ohlendorf, D. H., Takeda, Y., and Matthews, B. W. (1981) Nature 290, 754-758; Ohlendorf, D. H., Anderson, W. F., Fisher, R. G., Takeda, Y., and Matthews, B. W. (1982) Nature 298, 718-723). In the model, the carboxyl-terminal part of Cro was tentatively presumed to interact with the DNA minor groove. Protection of lysines 62 and 63 confirms the involvement of the carboxyl terminus in DNA binding. Although nonspecific and specific DNA protect the same lysine residues, there are differences in the nature of the interaction of Cro with nonspecific and specific DNA. Cro-nonspecific DNA interaction is salt-sensitive, suggesting that the interaction is predominantly electrostatic. On the other hand, Cro-specific DNA interaction is salt-resistant, suggesting that the interaction may include nonelectrostatic components (hydrogen bonds and hydrophobic interactions) as well. Protection experiments of tyrosine residues (against iodination) suggest that the conformation of Cro repressor changes in two stages: first, when Cro binds at nonspecific sites, and, second, when Cro binds to specific sites on DNA.     

1H NMR studies of lambda cro repressor. 2. Sequential resonance assignments of the 1H NMR spectrum

The cro repressor protein from bacteriophage lambda has been studied in solution by two-dimensional nuclear magnetic resonance spectroscopy (2D NMR). Following the approach of Wüthrich and co-workers [Wüthrich, K., Wider, G., Wagner, G., & Braun, W. (1982) J. Mol. Biol. 155, 311-319], individual spin systems were identified by J-correlated spectroscopy (COSY) supplemented, where necessary, by relayed coherence transfer spectroscopy (RELAY). Nuclear Overhauser effect spectroscopy (NOESY) was used to obtain sequence-specific assignments. From the two-dimensional spectra, the peptide backbone resonances (NH and C alpha H) for 65 of the 66 amino acids were assigned, as well as most of the side chain resonances. The chemical shifts for the assigned protons are reported at 35 degrees C in 10 mM potassium phosphate, pH 6.8, and in 10 mM potassium phosphate, pH 4.6, 0.2 M KCl, and 0.1 mM EDTA. Small shifts were observed for some resonances upon addition of salt, but no major changes in the spectrum were seen, indicating that no global structural change occurs between these ionic strengths. NOE patterns characteristic of alpha-helices, beta-strands, and turns are seen in various regions of the primary sequence. From the location of these regions the secondary structure of cro in solution appears to be virtually identical with the crystal structure [Anderson, W. F., Ohlendorf, D. H., Takeda, Y., & Matthews, B. W. (1981) Nature (London) 290, 754-758]. Missing assignments include the Pro-59 resonances and the peripheral protons of the eight lysine, the three arginine, and three of the five isoleucine residues.     

Lambda cro repressor complex with OR3 operator DNA. 19F nuclear magnetic resonance observations

The interaction of lambda cro repressor with DNA is probed using synthetic 17 base-pair OR3 operators in which 5-fluorodeoxyuridine has been systematically incorporated at each of the nine positions normally occupied by a thymidine residue. By monitoring changes in chemical shift of the fluorine resonances upon cro repressor binding in aqueous buffers of varying 2H2O content, we have examined the specific cro repressor-OR3 DNA complex in detail. The results are interpreted in the context of the popular model for cro repressor-OR3 complex derived from the three-dimensional structure of the cro repressor in the absence of DNA. The results presented here not originally predicted by the model are: (1) there is an asymmetry in the environment at the two ends of the operator, although the base-pairs involved and the cro repressor dimer are symmetric; (2) there appears to be distortion of the DNA helix at two distinct positions; (3) changes of the DNA environment in the middle of the helix suggest additional DNA distortion not near the contact areas proposed in the model.     

Substituting an alpha-helix switches the sequence-specific DNA interactions of a repressor

It has been suggested that many DNA-binding proteins use an alpha-helix for specific sequence recognition. We have used amino acid sequence homologies to identify the presumptive DNA-recognition helices in two related proteins whose structures are unknown--the repressor and cro protein of bacteriophage 434. The 434 repressor and cro protein each bind to three similar sites in the rightward phage 434 operator, OR, and they make different contacts in each binding site, as revealed by the chemical probe dimethyl sulfate. We substituted the putative recognition alpha-helix of 434 repressor with the putative recognition alpha-helix of 434 cro protein to create a hybrid protein named repressor*. The specific DNA contacts made by repressor* are like those of 434 cro protein.     

Recognition of DNA sequences by the repressor of bacteriophage 434

The structure of a complex between the DNA-binding domain of phage 434 repressor and a 14 base-pair synthetic DNA operator reveals the molecular interactions important for sequence-specific recognition. A set of contacts with DNA backbone, notably involving hydrogen bonds between peptide-NH groups and DNA phosphates, position the repressor and fix the DNA configuration. Direct interactions between amino acid side chains and DNA bases involve nonpolar van der Waals contacts as well as hydrogen bonds. The structures of the repressor domain and of the 434 cro protein are extremely similar. There appear to be no major conformational changes in the proteins when they bind to DNA.     

Binding and bending of the lambda replication origin by the phage O protein.

We have characterized the binding of lambda phage replication initiation protein O to the phage origin of replication. The minimal DNA segment required for O binding is the single iteron, a 19-bp sequence of hyphenated dyad symmetry that is repeated with variations four times in the origin. The isolated amino terminus of O protein is also sufficient to bind DNA. Electrophoretic studies show that the amino terminus of O protein induces bending of a single iteron. The DNA-protein interaction was characterized by ethylation interference, dimethyl sulfate protection and neocarzinostatin footprinting. Points of DNA-protein contact are largely concentrated in two areas symmetrically disposed with respect to the dyad symmetry of the iteron. This suggests the protein interacts as a dimer with half sites in the DNA. However, a few non-symmetrical contacts are found, indicating that O protein may distort the helix. This may correlate with the bending effects demonstrated electrophoretically. Cylindrical DNA projections were used to model O protein binding to the lambda origin and compare it with the lambda repressor-operator interaction. Whereas bound repressor nearly encircles the DNA in the major groove, O protein leaves the major groove on the opposite side exposed.     

Trigger effect in switching the phage lambda genome]

The paper presents a mathematical model of the trigger switching of lambda phage genome on the basis of the molecular ideas by Ptashne. The sigmoid character of the gene cro promotor was explained via lambda-repressor concentration. The temperature switching of genetic trigger was attributed to the existence of permissive and restrictive temperatures by means of the Fokker-Plank probability density formalism. The dependence of the lambda repressor average concentration of the UV-radiation dose was theoretically calculated. For the lambda phage mutant the hysteresic dependence of the lambda repressor and cro concentrations on the external parameters (for example, UV-intensity) was obtained.     

Interaction of lambda cro repressor with synthetic operator OR3 studied by competition binding with minor groove binders.

In the present work, we employ a combination of CD spectroscopy and gel retardation technique to characterize thermodynamically the binding of lambda phage cro repressor to a 17 base pair operator OR3. We have found that three minor groove-binding antibiotics, distamycin A, netropsin and sibiromycin, compete effectively with the cro for binding to the operator OR3. Among these antibiotics, sibiromycin binds covalently to DNA in the minor groove at the NH2 of guanine, whereas distamycin A and netropsin interact preferentially with runs of AT base pairs and avoid DNA regions containing guanine bases in the two polynucleotide strands. Only subtle DNA conformation changes are known to take place upon binding of these antibiotics. Both the CD spectral profiles and the results of the gel retardation experiments indicate that distamycin A and netropsin can displace cro repressor from the operator OR3. The binding of cro repressor to the OR3 is accompanied by considerable changes in CD in the far-UV region which appear to be attributed to a DNA-dependent structural transition in the protein. Spectral changes are also induced in the wavelength region of 270-290 nm. The CD spectral profile of the cro-OR3 mixture in the presence of distamycin A can be represented as a sum of the CD spectrum of the repressor-operator complex and spectrum of distamycin-DNA complex at the appropriate molar ratio of the bound antibiotic to the operator DNA (r). When r tends to the saturation level of binding the CD spectrum in the region of 270-360 nm approaches a CD pattern typical of complexes of the antibiotic with the free DNA oligomer. This suggests that simultaneous binding of cro repressor and distamycin A to the same DNA oligomer is not possible and that distamycin A and netropsin can be used to determine the equilibrium affinity constant of cro repressor to the synthetic operator from competition-type experiments. The binding constant of cro repressor to the OR3 is found to be (6 +/- 1).10(6)M-1 at 20 degrees C in 10 mM sodium cacodylate buffer (pH 7.0) in the presence of 0.1 M NH4F.     

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).     

DNA recognition by the helix-turn-helix motif: investigation by laser Raman spectroscopy of the phage lambda repressor and its interaction with operator sites OL1 and OR3.

The lambda repressor provides a model system for biophysical studies of DNA recognition by the helix-turn-helix motif. We describe laser Raman studies of the lambda operator sites OL1 and OR3 and their interaction with the DNA-binding domain of lambda repressor (residues 1-102). Raman spectra of the two DNA sites exhibit significant differences attributable to interstrand purine-purine steps that differ in the two oligonucleotides. Remarkably, the conformation of each operator is significantly and specifically altered by repressor binding. Protein recognition, which involves hydrogen-bond formation and hydrophobic contacts in the major groove, induces subtle changes in DNA Raman bands of interacting groups. These include (i) site-specific perturbations to backbone phosphodiester geometry at AT-rich domains, (ii) hydrophobic interaction at thymine 5CH3 groups, (iii) hydrogen bonding to guanine 7N and 6C = O acceptors, and (iv) alterations in sugar pucker within the C2'-endo (B-DNA) family. These perturbations differ between aqueous OL1 and OR3 complexes of repressor, indicating that protein binding in solution determines the precise DNA conformation. The overall structure of the lambda domain is not greatly perturbed by binding to either OL1 or OR3, in accord with X-ray studies of other complexes. However, Raman markers indicate a change in hydrogen bonding of the OH group of tyrosine-22, which is a hydrogen-bond acceptor in the absence of DNA but a combined donor and acceptor in the OL1 complex; yet, Y22 hydrogen bonding is not altered in forming the OR3 complex. The present results demonstrate qualitatively different and distinguishable modes of interaction of the lambda repressor DNA-binding domain with operators OL1 and OR3 in solution. This application of laser Raman spectroscopy to a well-characterized system provides a prototype for future Raman studies of other DNA-binding motifs under physiological conditions.     

Mutant λ Repressors with Increased Operator Affinities Reveal New, Specific Protein-DNA Contacts

The binding specificities of four mutant lambda cI repressor proteins with increased affinities for operator DNA were examined. Two mutant repressors (Glu34----Lys and Glu83----Lys) have the same specificity of binding as wild-type repressor, whereas two (Gly48----Ser and Gly48----Asn) have new binding specificities. The Gly48----Asn mutant repressor recognizes lambda operators with changes at base pair 3 with a different order of affinity than wild-type repressor, suggesting that the side chain of Asn48 makes additional specific DNA contacts at or near this base pair. When paired with a change that disrupts the specific interaction of the amino-terminal arm of lambda repressor with DNA (Lys4----Gln), one change that increases the affinity of repressor (Gly48----Ser) suppresses the binding defect of the Lys4----Gln repressor, resulting in a double mutant repressor with a new binding specificity different than that of both its parents and of wild type. These results lend strong support to the model of direct recognition of the lambda operator by lambda repressor proposed from the crystal structure of the repressor/operator complex.     

Comparison of the structures of cro and lambda repressor proteins from bacteriophage lambda

The three-dimensional structures of cro repressor protein and of the amino-terminal domain of lambda repressor protein, both from bacteriophage lambda, are compared. The second and third alpha-helices, alpha 2 and alpha 3, are shown to have essentially identical conformations in the two proteins, confirming the significance of the amino acid sequence homology previously noted between these and other DNA binding proteins in the region corresponding to these helices. The correspondence between the two-helical units in cro and lambda repressor protein is better than the striking agreement noted previously between two-helical units in cro and catabolite gene-activator protein. Parts of the first alpha-helices of repressor and cro show a structural correspondence that suggests a revised sequence homology between the two proteins in their extreme amino-terminal regions. In particular, there is a short loop between the alpha 1 and alpha 2 helices of lambda repressor that is missing from cro. This structural difference may account for the observed differences found with different cros and repressors in the pattern of phosphates whose ethylation prevents the binding of these proteins to their specific recognition sites. Although the two proteins have strikingly similar alpha 2-alpha 3 helical units that are presumed to bind to DNA in an essentially similar manner, stereochemical restrictions prevent the alpha 2-alpha 3 units of the respective proteins aligning on the DNA in exactly the same way.     

Coupled energetics of lambda cro repressor self-assembly and site-specific DNA operator binding II: cooperative interactions of cro dimers

The bacteriophage lambda relies on interactions of the cI and cro repressors which self assemble and bind the two operators (O(R) and O(L)) of the phage genome to control the lysogenic to lytic switch. While the self assembly and O(R) binding of cI have been investigated in detail, a more complete understanding of gene regulation by phage lambda also requires detailed knowledge of the role of cro repressor as it dimerizes and binds at O(R) sites. Since dimerization and operator binding are coupled processes, a full elucidation of the regulatory energetics in this system requires that the equilibrium constants for dimerization and cooperative binding be determined. The dimerization constant for cro has been measured as a prelude to these binding studies. Here, the energetics of cro binding to O(R) are evaluated using quantitative DNaseI footprint titration techniques. Binding data for wild-type and modified O(R) site combinations have been simultaneously analyzed in concert with the dimerization energetics to obtain both the intrinsic and cooperative DNA binding energies for cro with the three O(R) sites. Binding of cro dimers is strongest to O(R)3, then O(R)1 and lastly, O(R)2. Adjacently bound repressors exhibit positive cooperativity ranging from -0.6 to -1.0 kcal/mol. Implications of these, newly resolved, energetics are discussed in the framework of a dynamic model for gene regulation. This characterization of the DNA-binding properties of cro repressor establishes the foundation on which the system can be explored for other, more complex, regulatory elements such as cI-cro cooperativity.     

The missing nucleoside experiment: a new technique to study recognition of DNA by protein

We report a new technique for quickly determining which nucleosides in a DNA molecule are contacted by a sequence-specific DNA-binding protein. Our method is related to the recently reported "missing contact" experiment [Brunelle, A., & Schleif, R. F. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 6673-6679]. We treat the DNA molecule with the hydroxyl radical to randomly remove nucleosides. The ability of protein to bind to gapped DNA is assayed by gel mobility shift. Nucleosides important to protein binding are identified by sequencing gel electrophoresis. The missing nucleoside experiment can be used to scan a DNA molecule at single-nucleotide resolution in one experiment. The bacteriophage lambda repressor-OR1 and cro-OR1 complexes were analyzed to evaluate the method. For both proteins, the most important contacts are located in the protein monomer that binds to the consensus half of the operator. These contacts correspond well to those found by mutational studies, and in the cocrystal structure of the lambda repressor-operator. The missing nucleoside data show that the amino-terminal arms of lambda repressor make energetically important contacts with positions 7 and 8 and the central dyad base pair of the operator. The amino-terminal arm that makes the most extensive contacts to DNA appears to be the one that emanates from the repressor monomer that binds to the consensus half of the operator, in agreement with the cocrystal structure. The lambda cro protein does not have an amino-terminal arm, and the missing nucleoside experiment clearly shows a lack of contacts to DNA in the central region of the operator in this complex.     

Orientation of the Lac repressor DNA binding domain in complex with the left lac operator half site characterized by affinity cleaving.

Lac repressor (LacR) is a helix-turn-helix motif sequence-specific DNA binding protein. Based on proton NMR spectroscopic investigations, Kaptein and co-workers have proposed that the helix-turn-helix motif of LacR binds to DNA in an orientation opposite to that of the helix-turn-helix motifs of lambda repressor, lambda cro, 434 repressor, 434 cro, and CAP [Boelens, R., Scheek, R., van Boom, J. and Kaptein, R., J. Mol. Biol. 193, 1987, 213-216]. In the present work, we have determined the orientation of the helix-turn-helix motif of LacR in the LacR-DNA complex by the affinity cleaving method. The DNA cleaving moiety EDTA.Fe was attached to the N-terminus of a 56-residue synthetic protein corresponding to the DNA binding domain of LacR. We have formed the complex between the modified protein and the left DNA half site for LacR. The locations of the resulting DNA cleavage positions relative to the left DNA half site provide strong support for the proposal of Kaptein and co-workers.     

Design of the helix-turn-helix motif: nonlocal effects of quaternary structure in DNA recognition investigated by laser Raman spectroscopy

The operator-binding domain of phage lambda repressor provides a model for DNA recognition by the helix-turn-helix (HTH) motif. In the wild-type protein, dimerization is mediated by hydrophobic packing (of the dyad-related helix 5), which serves as an indirect determinant of operator affinity. The mutant repressor, Tyr88----Cys, forms an intersubunit disulfide linkage and exhibits enhancement of both structural stability and operator affinity. Yet the dimer-specific operator affinity of the mutant is 10-fold weaker than that of the wild-type (noncovalent) dimer, suggesting nonlocal effects of the intersubunit disulfide bond on HTH recognition (Sauer et al., 1986). To explore such nonlocal effects, we describe laser Raman studies of the Cys88 mutant repressor and its interaction with operator sites OL1 and OR3. The following results have been obtained: (i) Wild-type and mutant dimers exhibit similar secondary structures, indicated by quantitative comparison of Raman amide I and amide III bands. (ii) The engineered disulfide of the mutant lacks rigorous symmetry; we observe mainly the gauche/gauche/trans CC-S-S-CC rotamer. (iii) Remarkably, distinctive local and nonlocal differences are observed in the mechanisms of DNA recognition by wild-type and mutant repressors. These differences involve specific hydrogen-bonding interactions between the protein and DNA, including guanine N7 sites in the major groove of DNA, and alterations in DNA phosphodiester conformation induced by protein binding. We analyze these differences in relation to crystal structures of the wild-type dimer with and without bound DNA.(ABSTRACT TRUNCATED AT 250 WORDS)