Stability of the lac repressor headpiece against thermal denaturation and tryptic hydrolysis.

The stability of the conformation of the lac repressor headpiece against thermal denaturation and tryptic hydrolysis has been studied by circular dichroism measurements. In both cases the stability depends strongly on the concentration of NaCl. This effect is larger than generally observed for proteins. The midpoint of the thermal denaturation curve (Tm) is shifted from about 37 degrees C in the absence of NaCl to about 68 degrees C in 1 M NaCl. After a first non-linear increase of the Tm with the NaCl concentration (up to about 0.2 M NaCl) the Tm varies linearly with the salt concentration. Assuming a two-state mechanism for the thermal denaturation, enthalpies of 30-36 kcal/mol have been determined. The decrease of the circular dichroism signal due to the tryptic cleavage follows pseudo first-order kinetics for all salt concentrations studied. The half-life time of hydrolysis increased by about 40-times from 2 mM to the highest NaCl concentration we have used (655 mM). Assuming that only the unfolded state of the headpiece is a good substrate for trypsin, the observed stabilization against proteolytic degradation may be explained by a shift of the unfolding equilibrium of the headpiece due to the salt, and a subsequent decrease of the concentration of the unfolded state. The unusual stabilization of the headpiece is discussed with respect to its positive charge and to its function to bind to DNA.

LexA repressor induces operator-dependent DNA bending.

LexA, the repressor of the SOS system in Escherichia coli induces a substantial DNA bending upon interaction with the operator of the caa gene, which codes for the bacterial toxin colicin A. Analysis by gel electrophoresis of a family of DNA fragments of identical length, but bearing the caa operator at different positions, shows that DNA bending occurs close to or within the operator sequence upon LexA binding. In contrast, the interaction of LexA with the recA operator induces no detectable bending on 5% polyacrylamide gels. This difference between the two operators is likely to be due to an intrinsic bendability of the caa operator related to thymine tracts located on both sides of the operator. Such tracts do not exist in the recA operator. The free DNA fragments harbouring the caa operator show a slight tendency to bend even in the absence of the LexA repressor. The centre of this intrinsic bend is located close to or within the caa operator.

Isolation and characterization of LexA mutant repressors with enhanced DNA binding affinity.

The LexA repressor from Escherichia coli is a sequence-specific DNA binding protein that shows no pronounced sequence homology with any of the known structural motifs involved in DNA binding. Since little is known about how this protein interacts with DNA, we have selected and characterized a great number of intragenic, second-site mutations which restored at least partially the activity of LexA mutant repressors deficient in DNA binding. In 47 cases, the suppressor effect of these mutations was due to an Ind- phenotype leading presumably to a stabilization of the mutant protein. With one exception, these second-site mutations are all found in a small cluster (amino acid residues 80 to 85) including the LexA cleavage site between amino acid residues 84 and 85 and include both already known Ind- mutations as well as new variants like GN80, GS80, VL82 and AV84. The remaining 26 independently isolated second-site suppressor mutations all mapped within the amino-terminal DNA binding domain of LexA, at positions 22 (situated in the turn between helix 1 and helix 2) and positions 57, 59, 62, 71 and 73. These latter amino acid residues are all found beyond helix 3, in a region where we have previously identified a cluster of LexA (Def) mutant repressors. In several cases the parental LexA (Def) mutation has been removed by subcloning or site-directed mutagenesis. With one exception, these LexA variants show tighter in vivo repression than the LexA wild-type repressor. The most strongly improved variant (LexA EK71, i.e. Glu71----Lys) that shows an about threefold increased repression rate in vivo, was purified and its binding to a short consensus operator DNA fragment studied using a modified nitrocellulose filter binding assay. As expected from the in vivo data, LexA EK71 interacts more tightly with both operator and (more dramatically) with non-operator DNA. A determination of the equilibrium association constants of LexA EK71 and LexA wild-type as a function of monovalent salt concentration suggests that LexA EK71 might form an additional ionic interaction with operator DNA as compared to the LexA wild-type repressor. A comparison of the binding of LexA to a non-operator DNA fragment further shows that LexA interacts with the consensus operator very selectively with a specificity factor of Ks/Kns of 1.4 x 10(6) under near-physiological salt conditions.

Spacing requirements between LexA operator half-sites can be relaxed by fusing the LexA DNA binding domain with some alternative dimerization domains.

The dimerization domain of the LexA repressor has been replaced by two heterologous dimerization motifs: the "leucine zipper" from the jun oncogene product and the carboxy-terminal oligomerization domain of Escherichia coli lac repressor. The corresponding hybrid proteins LexA1-87-Jun zipper and LexA1-87-lac repressor have been purified and their DNA binding properties have been studied using gel mobility shift assays. Both fusion proteins form stable specific complexes with a short DNA duplex harboring the CTGT(at)4ACAG consensus sequence of the LexA repressor. This conserved DNA binding capacity distinguishes these two fusion proteins from many others containing a LexA DNA binding domain fused to different heterologous transactivation and/or dimerization domains. However the fusion proteins LexA1-87-Jun zipper and LexA1-87-lac repressor behave differently from native LexA repressor in that these fusion proteins tolerate the insertion of additional base-pairs between the two invertedly repeated CTGT motifs. LexA1-87-Jun zipper requires two CTGT motifs and tolerates the insertion of at least two additional base-pairs between these motifs, whereas LexA1-87-lac repressor requires in fact only a single CTGT motif for the formation of a specific complex detectable in gel mobility shift assays. The inability of the normal LexA repressor to form well-defined complexes with operators containing additional base-pairs in the center suggests that the LexA "hinge region" between the amino-terminal DNA binding and the carboxy-terminal dimerization domain might not be entirely flexible. In an attempt to remove a hypothetical interaction between the LexA cleavage site (which is situated within the hinge region) and the catalytic cleavage center (situated within the carboxy-terminal domain) a LexA mutant repressor containing five simultaneous mutations in the hinge region has been constructed and purified. Surprisingly this mutant repressor failed to form stable complexes detectable by the gel mobility shift assay even with the normal consensus sequence, suggesting that the LexA hinge region is more than a simple connector between the two structural domains and that its chemical nature is important not only for LexA cleavage, but also for the formation of stable LexA-DNA complexes.

Promoter properties and negative regulation of the uvrA gene by the LexA repressor and its amino-terminal DNA binding domain.

A comparative study of the interaction of the LexA repressor of Escherichia coli and of its amino-terminal DNA binding domain to the uvrA operator has been undertaken. Most of the binding constants are determined from competition experiments with RNA polymerase by measuring the time-course of the abortive initiation transcriptional activity. The presence of repressor increases the lag time, tau, without affecting the final maximum activity. The inhibition of transcription by LexA, at least in the case of the uvrA gene, is thus a transient, time-dependent phenomenon, because once the RNA polymerase is engaged in a stable "open" complex, it is quasi-irreversibly trapped in this state. A study of the binding constants as a function of ionic strength suggests the formation of 5.5(+/- 1) salt bridges between the uvrA operator and a LexA dimer. Surprisingly, the binding affinity of the amino-terminal domain was only about one order of magnitude smaller than that of the entire LexA repressor. The determination of the binding constant of the RNA polymerase to the "closed" uvrA promoter (KB approximately 1 X 10(7) to 2 X 10(7) M-1) allowed us to determine theoretical repression curves for the two repressor species. These calculations show that the binding constant found for LexA is sufficiently high to account for substantial or complete repression, and that of the amino-terminal domain is sufficiently low to account for partial or nearly full induction. Under solvent conditions used by others for the determination of binding constants to other SOS operators by DNAase I footprinting, the uvrA operator turns out to be a rather weak one (K approximately 3 X 10(7) M-1), being comparable with that of the uvrB gene. The uvrA promoter is "association-limited" with a KB X k2 product fitting very nicely the homology score for the promoter of 55.

Activation of recA protein. The open helix model for LexA cleavage.

RecA protein is induced by the binding of DNA and ATP to become active in the hydrolysis of ATP and the cleavage of repressors. These reactions appear to depend on the structural state of the protein polymerized along the DNA, i.e. a helical coat of six RecA per turn of 95 to 100 A pitch. In support of this model of the active conformation, it was shown that high concentrations of salt also induce this helical polymerized state as well as the enzymatic activities. Here, we describe that, in vitro and with the non-hydrolyzable analogue ATP gamma S, RNA and heparin can also induce both the structural transition and the enzymatic activation of RecA to LexA cleavage in accordance with the model. RNA and heparin do not support the reaction in the presence of ATP, and they do not induce the hydrolysis of ATP either, suggesting that, in contrast to ATP gamma S, the nucleotide is not bound stably enough, and that the combined affinities of polynucleotide and ATP actually modulate the discrimination of RecA for the various possible inducers in vivo.

Complexes of RecA protein in solution. A study by small angle neutron scattering.

RecA complexes on DNA and self-polymers were analysed by small-angle neutron scattering in solution. By Guinier analysis at small angles and by model analysis of a subsidiary peak at wider angles, we find that the filaments fall into two groups: the DNA complex in the presence of ATP gamma S, an open helix with pitch 95 A, a cross-sectional radius of gyration of 33 A and a mass per length of about six RecA units per turn, which corresponds to the state of active enzyme; and the compact form (bound to single-stranded DNA in the absence of ATP, or binding ATP gamma S in the absence of DNA, or just the protein on its own), a helical structure with pitch 70 A, cross-sectional radius of gyration 40 A and mass per length about five RecA units per turn, which corresponds to the conditions of inactive enzyme. The results are discussed in the perspective of unifying previous conflicting structural results obtained by electron microscopy.

Specificity of N-acetoxy-N-2-acetylaminofluorene-induced frameshift mutation spectrum in mismatch repair deficient Escherichia coli strains mutH, L, S and U.

The mismatch repair system of Escherichia coli is known to contribute to the fidelity of the replicational process. This system involves the functions of mutH, mutL, mutS and mutU (uvrD) loci which recognize mispaired bases as a consequence of errors due to the polymerase itself. Chemical modifications of DNA have also been suspected to create mispaired bases which, if the mispaired bases are removed, will lead to mutations by frameshift. Using the pBR322 plasmid DNA modified by the ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF) we have investigated this possibility in a forward mutational assay (tetracycline sensitivity). This fluorene derivative has been shown to induce predominantly frameshift mutations. Our results show that: The sensitivity of the deficient strains mutH, mutL and mutS to the AAF adducts is similar to that of the corresponding wild-type strain. However, the mutU strain appears much more sensitive to those adducts although less than a uvrA, B or C-deficient strain. This suggests that the mutU gene product is involved in the repair of AAF adducts. For the four mut deficient strains, and as it was shown with the wild-type strain, AAF adducts induced mutations to tetracycline sensitivity are only observed when the SOS system of the host bacteria is induced by irradiation of the cells prior to transformation with the modified plasmid. The mutation frequencies depend upon the ultraviolet light doses and similar maxima were found for the four mut strains and the corresponding wild-type strain. In agreement with the results obtained with wild-type or uvrA strains we observe that AAF adducts induce mostly frameshift mutations in the mut strains. Two types of hot spots of mutagenesis were described in wild-type and uvrA strains occurring either at repetitive sequences or at sequences of the type 5' G-G-C-G-C-C 3' (NarI restriction enzyme recognition sequence). While the second type of mutational hot spot does exist in the mismatch repair-deficient strains, we observe that the repetitive sequences are no longer hot spots of mutations in these strains, suggesting that the mismatch repair protein complex is involved in the establishment of AAF-induced frameshift mutations at repetitive sequences.

A LexA mutant repressor with a relaxed inter-domain linker.

The LexA protein is part of a large family of prokaryotic transcriptional repressors that contain an amino-terminal DNA binding domain and a carboxy-terminal dimerization domain. These domains are separated by a linker or hinge region, which is generally considered to be rather flexible and unconstrained. So far, no structure of any of the full-length repressors is available. Here we show that a mutant LexA repressor harboring several point mutations in the hinge region gets sensitive to trypsin and Glu-C cleavage over a segment of at least 20 amino acids, whereas the LexA wild-type hinge region is resistant to these proteases. These data are not compatible with the hypothesis of an fully flexible and/or unstructured inter-domain linker and suggest that the LexA hinge region is, in fact, constrained by contacts with the carboxy-terminal domain and/or a fairly stable local structure of the linker region.

Dorsal-B, a splice variant of the Drosophila factor Dorsal, is a novel Rel/NF-kappaB transcriptional activator.

The Drosophila transcription factor Dorsal, a member of the Rel/NF-kappaB family of proteins, plays a key role in the establishment of dorsoventral polarity in the early embryo and is also involved in the immune response. Here, we present evidence that the primary transcript of dorsal can be alternatively spliced, generating Dorsal-B, a new Rel/NF-kappaB family member. Dorsal and Dorsal-B are identical in the N-terminal region, which comprises both a DNA-binding domain and a dimerization domain. However, Dorsal-B lacks the nuclear localization signal located at the end of the Rel domain of Dorsal and is totally divergent in the C-terminal portion. Although Dorsal-B by itself is not able to induce the expression of a kappaB-controlled Luciferase reporter gene, we demonstrate that its C-terminal portion has transactivating properties. Analysis of the dorsal-B expression pattern indicates that the splicing is tissue-specific and excludes a putative role in early embryogenesis. However, dorsal-B synthesis is enhanced upon septic injury, and this challenge induces a nuclear accumulation of the protein in fat body cells suggesting that it may be involved in the immune response.

Cooperative and salt-resistant binding of lexA protein to non-operator DNA.

The interaction of the lexA repressor of E. coli with poly[d(A-T)] has been studied by circular dichroism. The binding induces an about 2-fold increase of the circular dichroism intensity at 263 nm, pointing out a conformational change of the nucleic acid. The observed spectral changes are very similar to those observed for the binding of the lac repressor to poly[d(A-T)] and natural DNA. At elevated ionic strength the binding isotherms do show a pronounced sigmoidal shape indicating a cooperative mode of binding.

A mutant LexA repressor harboring a cleavage motif cysteine-glycine remains inducible.

Using site-directed mutagenesis of the lexA gene we have changed the amino acid Ala-84 of the LexA repressor for a cysteine. The reason for this change was the striking homology between LexA and UmuD and the comparable size of the two amino acid side chains. Using an in vivo repression/induction assay it is shown that the LexA-Cys-84 mutant remains inducible by mitomycin C and UV irradiation essentially in the same way as the wild-type repressor.

Fluorescence study of the RecA-dependent proteolysis of LexA, the repressor of the SOS system in Escherichia coli.

The fluorescence of the LexA protein, the common repressor of the SOS system in Escherichia coli decreases by about 30% upon incubation with the RecA protein, and its cofactors ATP [or its non-hydrolysable analogue adenosine-5'-O-(3-thiotriphosphate), ATP gamma S] Mg2+ and single-stranded DNA. In the absence of any one of these elements required for the RecA-dependent proteolysis of LexA, this fluorescence change was not observed. The final fluorescence change depends only upon the concentration of LexA regardless of that of RecA. The time course of the fluorescence decrease corresponds well with the kinetics of the decrease of intact LexA protein and the increase of its 2 proteolytic fragments as determined by SDS-polyacrylamide gel electrophoresis. These results allow us to use the fluorescence change as a signal for a detailed kinetic analysis. The velocity of the proteolysis (d[LexA]/dt) is proportional to the concentration of LexA and RecA indicating that the formation of the LexA-RecA complex is the limiting step.

The carboxy-terminal domain of the LexA repressor oligomerises essentially as the entire protein.

The ability of the isolated carboxy-terminal domain of the LexA repressor of Escherichia coli to form dimers and tetramers has been investigated by equilibrium ultracentrifugation. This domain, that comprises the amino acids 85-202, is readily purified after self-cleavage of the LexA repressor at alkaline pH. It turns out that the carboxy-terminal domain forms dimers and tetramers essentially as the entire LexA repressor. The corresponding association constants were determined after non-linear least squares fitting of the experimental concentration distribution. A dimer association constant of K2 = 3 X 10(4) M-1 and a tetramer association constant of K4 = 2 X 10(4) M-1 have been determined. Similar measurements on the entire LexA repressor [(1985) Biochemistry 24, 2812-2818] gave values of K2 = 2.1 X 10(4) M-1 and K4 = 7.7 X 10(4) M-1. Within experimental error the dimer formation constant of the carboxy-terminal domain may be considered to be the same as that of the entire repressor whereas the isolated domain forms tetramers slightly less efficiently. It should be stressed that the potential error in K4 is higher than that in K2. The overall conclusion is that the two structural domains of LexA have also well-defined functional roles: the amino-terminal domain interacts with DNA and the carboxy-terminal domain is involved in dimerisation reinforcing in this way the binding of the LexA repressor to operator DNA.

In vitro study of the interaction of the LexA repressor and the UvrC protein with a uvrC regulatory region.

The in vitro interaction of the LexA repressor with a regulatory region of the uvrC gene has been studied by polyacrylamide gel electrophoresis. Although the uvrC promoter region shows some homology with the canonic LexA binding site, no specific binding of the repressor to this DNA sequence could be observed, but only a cooperative nonspecific binding. By the same technique we show that the UvrC protein does not bind specifically to this regulatory DNA sequence either, although the protein is able to bind nonspecifically and cooperatively to the double-stranded DNA fragment.

Fast abortive initiation of uvrA promoter in a supercoiled plasmid studied by stopped-flow techniques.

In order to follow the fast kinetics of abortive initiation (lag time from 1 ms to 10 s), we have built a stopped-flow apparatus equipped for fluorescence detection. The small volume used for each assay (35 microliters), and the short dead time (approximately 0.5 ms) are the essential advantages of this apparatus. Supercoiling of DNA affects considerably the initiation of transcription from the uvrA promoter. It decreases the lag time due to the isomerisation process 3-fold. Nevertheless, it does not change significantly the product KBk2, which is indicative of promoter strength and shows that uvrA is an 'association-limited' promoter. The presence of the LexA repressor increases the lag time considerably. At least for small RNA polymerase concentrations this increase is stronger for supercoiled than for linearized DNA.

DNA binding properties of the LexA repressor.

The LexA repressor from Escherichia coli negatively regulates the transcription of about 20 different genes upon binding with variable affinity to single-, double- or even triple-operators as in the case of the recN gene. Binding of LexA to multiple operators is cooperative if the spacing between these operators is favorable. LexA recognizes DNA via its amino-terminal domain. The three-dimensional structure of this domain has been determined by NMR measurements. It contains three alpha-helices spanning residues 8-20, 28-35 and 41-54. In view of this structure, but also according to homology considerations and the unusual contact pattern with the DNA backbone, the LexA repressor is not a normal helix-turn-helix DNA binding protein like for example phage lambda repressor. LexA is at best a distant relative of this class of transcription factors and should probably be considered as a protein that contains a new DNA binding motif. A cluster of LexA mutant repressors deficient in DNA binding falling into the third helix (residues 41-54 bp) suggests that this helix is involved in DNA recognition.

1H-NMR investigation of the interaction of the amino terminal domain of the LexA repressor with a synthetic half-operator.

A synthetic half-operator DNA-duplex, d(GCTACTGTATGT), containing a portion of the proposed recognition sequence (CTGT) of several "SOS" genes, has been synthesized. The dodecamer has been characterized through 1H-NMR spectroscopy. Complete assignment of exchangeable hydrogen bonded imino protons has been achieved by applying 1D NOE techniques and an analysis of the temperature dependence of the chemical shifts. In order to determine the specific role of the CTGT consensus sequence in the overall recognition process, the oligonucleotide duplex has been titrated with the amino terminal DNA binding domain of the LexA repressor. The observation of substantial changes of 1H-NMR chemical shifts in the imino proton region upon interaction with the protein strongly suggests that the protein binds specifically to the operator DNA. The largest deviations of 1H-NMR chemical shifts upon protein binding have been observed for protons assigned to the CTGT segment, thus strongly suggesting a direct involvement of this sequence in the binding process. At high potassium chloride concentrations the 1H-NMR chemical shift deviations are reverted which is consistent with the known drop in the affinity constant of LexA for operator DNA at high salt concentrations.

Investigation of RecA--polynucleotide interactions from the measurement of LexA repressor cleavage kinetics. Presence of different types of complex.

The proteolysis of the LexA repressor in the presence of RecA and various polynucleotides was studied by measuring the fluorescence decrease of LexA upon cleavage. The results were compared with the DNA binding of RecA to investigate the presence of multiple DNA-RecA complexes. All single-stranded polydeoxyribonucleotides (DNA) efficiently stimulated the proteolysis and the maximum activation was reached in the presence of three or four nucleotides of polynucleotide per monomer of RecA. The stimulative effect was decreased in the presence of larger amounts of poly(dA), poly(dT) or heat-denatured DNA, whereas the excess of single-stranded DNAs chemically modified with chloroacetaldehyde did not present such an inhibitory effect, despite the fact that a second DNA molecule is likely to interact with RecA as monitored by the intrinsic fluorescence of these DNA species. The complicated cleavage promotion and inhibition pattern is tentatively explained by a three-state model assuming that RecA may interact with three single-stranded DNA molecules. According to this model, occupation of the first site would be necessary and sufficient for cleavage promotion, the second site would be neutral with respect to cleavage and the occupation of the third site would inhibit LexA cleavage at least partially. Double-stranded natural DNA did not stimulate cleavage, even under conditions where RecA binds quantitatively to the DNA. No polyribonucleotides (RNA) examined showed a significant stimulative effect either, nor did they appear to interact with RecA.

Contacts between the LexA repressor--or its DNA-binding domain--and the backbone of the recA operator DNA.

Using hydroxyl radical footprinting and ethylation interference experiments, we have determined the backbone contacts made by the entire LexA repressor and its amino-terminal fragment with the recA operator DNA. These techniques reveal essentially the same contacts between both proteins and one side of the DNA helix if one assumes that the DNA stays in the normal B-conformation. This result is somewhat unexpected because protection of guanine bases against methylation suggested a somewhat twisted recognition surface. The backbone contacts revealed by both methods are symmetrically disposed with respect to the center of the operator, providing further evidence that the operator binds two LexA monomers. Each half-operator contains seven interfering phosphates. These phosphates are found on both sides of the 5'-CTGT sequence that is believed to be the principal recognition target. On the side close to the center of the operator are found two phosphates, whereas the other five are clustered on the side apart from the dyad axis. We are not aware of such an extended cluster of interfering phosphates for any other DNA-binding protein. A quantification of the hydroxyl radical footprints allowed us to compare further the affinity of the LexA repressor for the recA operator with that of its isolated DNA binding domain. We find an only 13-fold higher binding constant for LexA than for its amino-terminal domain, which is in good agreement with our earlier results for the uvrA operator using a completely different binding assay.