Repressor-operator interaction in the lac operon: III. Nuclear magnetic resonance observations with altered amino-terminal DNA binding domains

We have examined the N-terminal 56 amino acid fragment, the domain that can bind DNA independently, from 3-fluorotyrosine-substituted Escherichia coli lac repressor by ¹⁹F-nuclear magnetic resonance. The fragments or “headpieces” from four altered repressers missing each of the tyrosines in turn were examined in parallel. When the wild-type N-terminal fragment is titrated with a 36 base-pair lac operator DNA sequence, the ¹⁹F resonances undergo changes in their chemical shifts that are different from those changes when the N-terminal fragment is titrated with non-specific DNA fragments. By looking at these operator-induced changes as well as pH-dependent effects with all four altered N-terminal fragments, we show systematic correlations with the genetic data. The data lead us to conclude that upon operator DNA binding: (1) tyrosine 7 is displaced to a less polar environment and the higher than normal pK value of the phenolic OH group is decreased; (2) tyrosine 12 does not change much in either its mobility or environment; and (3) tyrosine 17 is involved, as suggested by the genetic data, when the headpiece forms a complex with operator DNA.     

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.     

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.     

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.     

Stereospecific assignments of 1H-nmr methyl lines and conformation of valyl residues in the lac repressor headpiece

Two-dimensional ¹H-nmr methods are described to obtain information on the sidechain conformation of valyl residues of the lac repressor headpiece and to assign the resonances of their methyl groups stereospecifically. The spin–spin coupling constants (J_αβ) between C^αand C^β protons are obtained from two-dimensional correlated spectroscopy experiments. Large values for J_αβ(10–12 Hz) corresponding to trans orientations for these protons (g⁺ conformation) are found for all valyl residues in α-helical segments. For these valyl residues, the distance between one methyl group (γ₁)and the valyl amide proton is much shorter than for the other methyl group, so that stereospecific resonance assignments follow from relative intensities of the corresponding cross peaks in a two-dimensional nuclear Overhauser enhancement spectrum. Thus, streospecific assignments could be made for the methyl groups of Val 9, 20, 23, and 38 (of a total of eight valyl residues).     

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.     

Complexes of Escherichia coli lac-repressor with non-operator DNA revealed by electron microscopy: two repressor molecules can share the same segment of DNA.

Complexes of Escherichia coli lac-repressor with non-operator DNA have been visualized in the electron microscope using high-resolution metal shadowing and negative staining. Under conditions of a high ratio of repressor to DNA, all the DNA molecules are covered by repressor molecules and the resulting complexes appear as flattened ribbons with a width of approximately 200 Å. The overall dimensions of these complexes and their substructure indicate that it is very likely that repressor molecules are tightly packed on both “sides” of the DNA helix. Thus two repressor molecules can share the same segment of non-operator DNA by binding to opposite sides of the DNA helix.     

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.     

Dielectric measurements on live biological materials under magnetic resonance conditions

In the course of work on the interactions of electric and magnetic fields with both living and dead biological materials, it was noticed that certain published dielectrophoretic yield curves for biological cells showed unexplained deviations in the region of 2 kHz. Dielectrophoretic measurements made at frequencies and magnetic fields which satisfied the nuclear magnetic resonance conditions showed sharply resonant features. Dielectric measurements showed small, but sharp, resonances most easily seen in the dielectric loss curves which had a bandwidth of the order of one Hertz and presented at the frequencies which satisfied the magnetic resonance conditions for the ambient magnetic field. Resonances were found corresponding to the frequencies for electron spin resonance and nuclear magnetic resonance for¹H,³¹P,²³Na,³⁷Cl and³⁹K. The onset of these resonances occurs at the value of the steady magnetic field strength so that one quantum of magnetic flux (2.07×10^?15wb) would link a single biological cell or pair of cells, approximately 1 G (100μT) in the case of a 5-μm yeast cell. The effects of these magnetic resonance conditions on the mean generation time ofE. coli and on the reaction of the enzyme lysozyme with the substrateM. lysodeikticus cells are also shown.     

High resolution nuclear magnetic resonance studies of the active site of chymotrypsin. I. The hydrogen bonded protons of the "charge relay" system

High resolution proton nuclear magnetic resonance has been used to observe protons at the active site of chymotrypsin A_δ and at the same region of chymotrypsinogen A. A single resonance with the intensity of one proton is located in the low field region of the nuclear magnetic resonance spectrum. This resonance is observed in H₂O solutions but not in ²H₂O. On going from low to high pH the resonance titrates upfield 3 parts per million in both proteins and has a pK of 7.5. The titration can be prevented by alkylating His57 with either of two active site directed chloromethyl ketones. Using these data the proton resonance has been assigned to a proton in a hydrogen bond between His57 and Asp102. Further confirmation of this assignment lies in the observation of a similar resonance in this same low field region of the nuclear magnetic resonance spectrum of trypsin, trypsinogen, subtilisin BPN′ and α-lytic protease all of which have the Asp-His-Ser triad at their active sites.This proton resonance in chymotrypsin A_δ was used as a probe to monitor the charge state of the active site upon formation of a stable acyl-enzyme analogue N²(N-acetylalanyl)-N¹benzoylcarbazoyl-chymotrypsin A_δ. In this derivative the His-Asp proton resonance titrates from the same low pH end point as in the native enzyme, ?18 parts per million, to a new high pH end point of ?14.4 parts per million (versus ?15.0 parts per million in the native enzyme). The difference of 0.6 parts per million in the high pH end points between the native and acyl enzyme is interpreted as supporting the suggestion that a hydrogen bond exists between Ser195 and His57 in the native enzyme and zymogen.We conclude from these studies that the charge relay system from Asp102 across His57 to Ser195 is intact in chymotrypsin A_δ and chymotrypsinogen A, and that, in the native enzyme, it slightly polarizes Ser195.     

A new class of promoter mutations in the lactose operon of Escherichia coli

The isolation and genetic characterization of a number of mutations that are located in the promoter region of the lac2 operon are described. These mutations have reduced levels of lac operon expression in a wild, type (crp⁺cya⁺) genetic background. Three of the mutations also have lower levels of lac operon expression than lacP⁺ in a crp^?cya^? genetic background, that is in the absence of the catabolite activator protein and 3′,5′-adenosine cyclic monophosphate. These three mutations are located nearest to the lac operator. They define a second essential site in the promoter region.     

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.     

Regeneration in Alfalfa Tissue Culture: Characterization of Intracellular pH During Somatic Embryo Production by Solid-State P-31 NMR

The composition of phosphorus-containing compounds of intact lyophilized alfalfa tissue has been determined, in part, by solid-state ³¹P nuclear magnetic resonance. The tissue (Medicago sativa L., cv Regen S; and some of its crosses) was grown in culture under both nonregenerating and regenerating conditions, the latter enhanced by the addition of specific amino acids. Analysis of the ³¹P nuclear magnetic resonance spectra shows that regeneration is favored when metabolism occurs without the production of a low average intracellular pH.     

Determination of the mode of interaction of Mn2+ and Cu2+ with cis-inositol by 13C NMR spectroscopy

Natural abundance ¹³C nuclear magnetic resonance spectroscopy (¹³C NMR) was used to study the mode of binding of Mn²⁺ and Cu²⁺ to the cyclitol, cis-inositol. Resonance linewidths and the electron nuclear relaxation rates [(T₁^e)^?1 values] were used to establish that a unique binding site exists for these metal-ions on this cyclitol involving only the three axial hydroxyl groups. This work may aid in the development of new organometallic complexes used as paramagnetic relaxation agents in magnetic resonance imaging research.     

Novel Mechanism of Regulation of Protein 4.1G Binding Properties Through Ca2+/Calmodulin-Mediated Structural Changes

Protein 4.1G (4.1G) is a widely expressed member of the protein 4.1 family of membrane skeletal proteins. We have previously reported that Ca²⁺-saturated calmodulin (Ca²⁺/CaM) modulates 4.1G interactions with transmembrane and membrane-associated proteins through binding to Four.one-ezrin–radixin–moesin (4.1G FERM) domain and N-terminal headpiece region (GHP). Here we identify a novel mechanism of Ca²⁺/CaM-mediated regulation of 4.1G interactions using a combination of small-angle X-ray scattering, nuclear magnetic resonance spectroscopy, and circular dichroism spectroscopy analyses. We document that GHP intrinsically disordered coiled structure switches to a stable compact structure upon binding of Ca²⁺/CaM. This dramatic conformational change of GHP inhibits in turn 4.1G FERM domain interactions due to steric hindrance. Based upon sequence homologies with the Ca²⁺/CaM-binding motif in protein 4.1R headpiece region, we establish that the 4.1G S⁷¹RGISRFIPPWLKKQKS peptide (pepG) mediates Ca²⁺/CaM binding. As observed for GHP, the random coiled structure of pepG changes to a relaxed globular shape upon complex formation with Ca²⁺/CaM. The resilient coiled structure of pepG, maintained even in the presence of trifluoroethanol, singles it out from any previously published CaM-binding peptide. Taken together, these results show that Ca²⁺/CaM binding to GHP, and more specifically to pepG, has profound effects on other functional domains of 4.1G.     

Specialized Transduction with λplac5: Involvement of the Rece and Recf Recombination Pathways

Several aspects of the recombination resulting from λ plac5 transduction were investigated in strains of Escherichia coli K-12 that use the RecE or RecF recombination pathways. In a RecBC pathway strain, F42lac recombination with λplac5 is 20- to 50-fold higher than chromosomal lac times λplac5 recombination, and this recombination enhancement is largely dependent on constitutive expression of F42lac fertility functions. Here, it was observed that F42 lac fertility functions do not effect the ability of F42lac to recombine with λplac5 in a RecE or RecF pathway strain. Therefore, the enhancement observed in a Rec⁺ (or RecBC pathway) strain is directly dependent on the recBC gene product. The end product of recombination between λplac5 and either F42lac or chromosomal lac in RecE and RecF pathway strains was monitored by scoring for addition and substitution transductants. It was observed that the percentage of addition transductants was lower in all cases for RecE and RecF pathway strains as compared with RecBC pathway or a recB strain. It is concluded that the introduction of sbcA or sbcB into a recB strain produces a change in recombination mechanism that is reflected in the nature of the end product of recombination.     

The spread of plasmids in model populations of Escherichia coli K12.

Comparison of R100 with its derepressed derivative R100-1 showed that the capacity to repress tra function does not significantly affect the spread by retransfer of R100. F′lac was used to investigate the contributions of growth and transfer to spread of a plasmid through a recipient population. Ability to transfer F′lac was lost rapidly when donor cultures entered stationary phase, but aggregate-forming ability was lost much more slowly. Comparison of F′lactra⁺ with F′lactraH88, which is unable to retransfer from recipients, showed the importance of retransfer. We used a mathematical model to calculate the amount of retransfer needed to explain the rate of increase of F′lac progeny. This showed that the lag between a cell receiving F′lac and being able to retransfer it was a less important constraint on this rate of increase than the inherent rate of plasmid transfer by established donors.     

Host range of conjugation and replication functions of the Escherichia coli sex plasmid Flac. Comparison with the broad host-range plasmid RK2

The Escherichia coli conjugative plasmid Flac has a restricted host range, in that transfer to Pseudomonas aeruginosa is not detectable. The molecular basis for this host-range restriction was studied by a separate comparison of the replication and conjugation systems of Flac with those of the broad host-range plasmid RK2. The origin of transfer of Flac (oriT_F) was cloned onto a small RK2 replicon. The hybrid plasmid, pDG2906, could be transferred efficiently by both the Flac and RK2 conjugation systems to an E. coli recipient. The Flac conjugation system was able to transfer pDG2906 to P. aeruginosa, but only at a frequency of 10^?4 of that of the RK2 conjugation system. A second hybrid plasmid, containing the replication region of Flac with the transfer region of RK2, could not be established in P. aeruginosa. These results show that Flac is able to mediate low frequency transfer to P. aeruginosa, and that the lack of replication in Pseudomonas is ultimately responsible for the restricted host range.     

Mobility of elastin chains as determined by 13C nuclear magnetic resonance

Relaxation times and integrated intensities of ¹³C have been obtained from nuclear magnetic resonance spectra of elastin in unstretched calf ligamentum nuchae and indicate that about 80% of the backbone carbonyl carbons have short rotational correlation times, τ_R ～ 40 nanoseconds. τ_R is reduced by only a factor of two when the ligament is in contact with 2 m-KCNS, a strong denaturant. By contrast, the highly ordered chains of collagen in insoluble calf achilles tendon give no spectrum until denatured in 2 m-KCNS, when t_R decreases by many orders of magnitude. These results show that elastin is composed largely of highly mobile chains under physiological conditions, suggesting that configurational entropy has an important role in its elastic properties.