©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
How 434 Repressor Discriminates Between O1 and O3
THE INFLUENCE OF CONTACTED AND NONCONTACTED BASE PAIRS (*)

(Received for publication, September 16, 1994; and in revised form, October 9, 1994)

Adam C. Bell (§) Gerald B. Koudelka (¶)

From the Department of Biological Sciences, State University of New York, Buffalo, New York 14260

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The sequence of the bacteriophage 434 O(R)1 (ACAAAACTTTCTTGT) differs from its O(R)3 (ACAGTTTTTCTTGT) at positions 4-6. X-ray analysis shows that the side chain of Gln of the 434 repressor makes van der Waals' and H-bond contacts with the T at position 4` in complex with O(R)1, but no specific contact is observed at this position in 434 repressor-O(R)3 complexes. No contacts are made by repressor to the bases at positions 5 or 6 in either binding site. The significance of the sequence differences between O(R)1 and O(R)3 in determining the operator affinity for repressor were examined by constructing synthetic variants of these operators. Measurements of the affinity of these operators for repressor as a function of ionic strength revealed that although base pairs 5 and 6 are not contacted by 434 repressor, they can nonetheless influence operator affinity for repressor by modulating the degree to which ionic interactions contribute to the overall binding energy. Both the magnitude and direction of their effect depends on the status of repressor's contacts to the bases at position 4. The role of contact made by Gln to position 4 was examined by mutating this amino acid to Ala and by examining the affinity of wild type repressor for an operator bearing a 5-methylcytosine at position 4` in an O(R)1-4G mutant. These experiments showed that repressor's preferences at operator positions 5 and 6 are linked to its position 4 preference via a van der Waals' contact between amino acid 33 and a methyl group on the base at operator position 4`. Together, the results of the experiments shown here reveal that bases that do not contact the protein alter its preferences for bases at the contacted operator position 4.


INTRODUCTION

Repressor is a DNA binding protein encoded by the bacteriophage 434. Transcription of the genes that define the developmental fate of the phage in Escherichia coli is controlled, in part, by the binding of 434 repressor to six binding sites on the bacteriophage chromosome. These six binding sites are divided between two operator regions termed O(R) and O(L). Repressor's discrimination between two of these binding sites, O(R)1 and O(R)3, provides a critical hinge point in determining the transcriptional activity at O(R) and, therefore, the fate of the phage in the cell(1, 2) .

Similar to the repressors of other lambdoid phages (1, 2) 434 repressor protein binds to DNA as a dimer. Each monomer of the protein is divided into two functionally distinct domains(3) . The carboxyl-terminal domain (amino acids 71-209) is responsible for dimerization of the intact repressor, while within R1-69, the amino-terminal domain (amino acids 1-69) residues in a helix-turn-helix motif determine DNA binding specificity(4) . Fig. 1shows the sequences of O(R)1 and O(R)3 and two position 4 variants of those operators. X-ray structural investigations show that the 434 repressor only makes specific contacts to the symmetrically arrayed outer four base pairs (positions 1-4 and 11-14 in Fig. 1) in these pseudosymmetric binding sites(5, 6) . Elimination of these contacts by mutation at these bases abolishes specific complex formation(7, 8) . Similar to the case with repressor(9) , changes in the central 6 bases in the 434 operator (5-10 in Fig. 1) dramatically alter operator affinity for 434 repressor(10, 11) . Unlike the case, however, no contacts are made between the 434 repressor and the central 6 base pairs of the 434 operator(5, 12) . We have shown that the central base pairs in the 434 operators influence the affinities of operators for 434 repressor by altering the structural deformability of the DNA binding sites(6, 11) . Our previous work in the showed that the differing sequences in the noncontacted regions of O(R)1 and O(R)3 alters 434 repressor's ability to discrimination between different bases at a contacted position of these operators, presumably by modifying the structure of the operator in the repressor-operator complexes(13) . No similar effects of sequence-dependent differences in DNA structure on sequence recognition by proteins have yet been reported.


Figure 1: 434 operator sequences. Sequence of O(R)1 and O(R)3 and the respective position 4 mutants used in this study. The base pair at position 4 in the mutants is circled. Throughout this paper, operators are named by their wild-type counterpart with the base at positions 4, 5, and/or 6 indicated where appropriate.



Analysis of the crystal structure of R1-69 bound to O(R)1 indicates that glutamine 33 of repressor makes a hydrogen bond to the O-4 of the thymine at position 4` and a van der Waals' contact with the methyl group on the same base(5, 6) . As a result of the base sequence difference at position 4 in O(R)3, repressor can not make either contact to that base when bound to O(R)3 ((6) , see Fig. 1). Since this AbulletT GbulletC substitution at position 4 is the only sequence difference within the contacted base pairs of O(R)1 and O(R)3, the loss of contacts at position 4 is thought to be the primary determinant of the 6-8-fold lower affinity of repressor for O(R)3(6, 10, 13, 14) . In agreement with this, we showed that swapping the base pair at position 4 between O(R)1 and O(R)3 reverses their order of affinity for repressor(13) . Our earlier results show, however, that the energetic cost of an AbulletT GbulletC change at position 4 is greater in O(R)1 than O(R)3(13) . Thus, the presence or absence of the contact between repressor and position 4` is not the only determinant of the difference in repressor's affinities for O(R)1 and O(R)3. Biochemical characterization of the complexes formed between repressor and these operators revealed that the ionic contribution to the affinity of repressor for these operators is not solely determined by the identity of the base pair at position 4. Instead, specific position 4/central sequence combinations direct the ionic strength dependence and affinity order. Together these results indicated that the loss of a contact, due to base pair substitution at position 4, is only partly responsible for the affinity difference between O(R)1 and O(R)3. Indeed, these results suggest that sequence dependent variation in the strength or number of ionic contacts between protein and DNA phosphate backbone may have a role in modulating specific base recognition by 434 repressor.

Considering the sequences of O(R)1 and O(R)3 in light of these data reveals that the degree to which an AbulletT GbulletC change at position 4 affects the affinity of repressor must be modulated by the identity of the base pairs at positions 5 and/or 6 (see Fig. 1). In this paper we report the results of experiments that directly test the role of the base pairs at positions 4-6 in determining the degree to which 434 repressor discriminates between O(R)1 and O(R)3. The change in operator affinity for repressor in response to a particular mutation at position 4 depends critically on the identity of the noncontacted base pairs at positions 5 and 6. The identity of each of these base pairs has a dramatic influence on the contribution of ionic interactions to the affinity of repressor for these operators. The results of other experiments demonstrate that a contact between the amino acid at position 33 of repressor and a 5-methyl group on a pyrimidine at operator position 4` is critical to the affinity of operator for repressor and plays a pivotal role in determining the ionic strength sensitivity of repressor's affinity for position 4 variants of O(R)1.


EXPERIMENTAL PROCEDURES

Binding Sites and Plasmids

DNA manipulations were performed as described(15) . Binding site oligonucleotides, except for O(R)1-4GbulletC, (^1)were synthesized on an Applied Biosystems model 380A DNA synthesizer, phosphorylated, annealed, and ligated into the unique SalI site in pUC18(16) . The sequence of the resulting clones was determined by dideoxy sequencing methods. The C-containing oligonucleotide and its complement were obtained from International DNA Technologies (Coralville, IA) and used directly in binding experiments (see below).

Protein Preparation

Wild-type and Ala repressor protein were isolated from E. coli strain X90 bearing plasmids that directed overproduction of 434 repressor or a position 33 glutaminealanine mutant(17) . Both proteins were purified according to the procedures in Wharton(14) .

Determination of Dissociation Constants

All dissociation constants are defined as the concentration of repressor monomer needed to give a fractional saturation of 0.5 for each operator studied. For wild-type 434 repressor-operator complexes, except those with O(R)1-4GbulletC, the dissociation constants were determined by a nitrocellulose filter binding assay as described in (4) . Operator-containing plasmids were linearized by cleavage at their unique with EcoRI site; the recessed ends were repaired with the Klenow fragment of DNA polymerase I in the presence of [alpha-P]dATP. The resulting 2700 base pair linear DNAs were used directly in filter binding experiments at concentrations less than 0.1 nM. To measure dissociation constants, these DNAs were incubated for 10 min at 23 °C with varying amounts of repressor in a buffer containing 10 mM TrisbulletHCl (pH 7.4), 100 µg ml bovine serum albumin, 1 mM dithiothreitol, and 50 or 100 mM KCl. The counts retained on the filter as a function of 434 repressor concentration were converted to fractional saturation values(18) . Fractional saturation values were determined from duplicate measurements. At least three duplicate measurements were averaged, and dissociation constants were determined from a nonlinear least squares fit to those data. In these and all other experiments, the protein concentrations cited are corrected for activity (50% of protein is active in DNA binding). The standard deviations of the dissociation constants obtained in this manner are ± 15%.

Complexes between operator and Ala-mutant repressor are unstable to filtration. Thus, in all experiments involving Ala, the dissociation constants were determined by quantitative nuclease protection assays analogous to those described by (19) , but using Cu(I)-phenanthroline as the nuclease. Cu(I)-phenanthroline protection assays were performed essentially as described by (20) . Briefly, 3`-end labeled operator-containing fragments were generated by cleavage of operator-containing plasmids with HindIII and HaeIII and repair of the recessed HindIII ends in the presence of [alpha-P]dATP. This protocol generates a labeled 260-base pair operator-containing fragment and a 14-base pair labeled fragment that does not interfere with cleavage analysis. To measure ionic strength dependence, these DNA fragments were incubated with varying amounts of mutant repressor in buffer containing <0.5 nM DNA, 10 mM TrisbulletHCl (pH 7.4), 100 µg ml bovine serum albumin, and 50 or 100 mM KCl. After a 10-min incubation at 23 °C, sufficient Cu(I)-phenanthroline was added to give, on average, one cleavage/DNA molecule in 4 min of further incubation. Following incubation with the nuclease, the DNAs were extracted with phenol/chloroform (50:50 (v/v)), precipitated with ethanol, suspended in 90% formamide dye mix, and denatured by heating at 90 °C. The samples were electrophoresed on 25 times 30 cm, denaturing, 7.5% polyacrylamide gels containing 7.5 M urea, 89 mM Tris borate (pH 8.9) and 1 mM EDTA. Autoradiography was performed by exposing the gel to preflashed Kodak XAR-5 film with an intensifying screen at -80 °C. Band intensities of the footprinting results were quantified, and the dissociation constants were determined from at least four different autoradiograms generated from separate experiments.

Repressor-O(R)1-4GbulletC complexes are not stable to either filtration or Cu(I)-phenanthroline digestion, hence quantitative DNase I footprinting was used to determine the affinity of 434 repressor for O(R)1-4GbulletC, essentially as described(19) . To generate the O(R)1-4GbulletC operator, a 30-base oligonucleotide of the sequence 5`-GGGGTCGAACAAGAAAGTCTGTTCGACCCC-3` and its complement were subjected to electrophoresis through a denaturing 20% polyacrylamide gel and purified on DEAE paper according to the manufacturer's instructions (Schleicher & Schuell). One strand was then 5`-end labeled with [-P]ATP and polynucleotide kinase, annealed to its complement, and the resulting double-stranded product was purified on a nondenaturing 20% polyacrylamide gel. Following further purification on DEAE paper, this 30-base pair operator-containing duplex was used in DNase I footprinting experiments. Binding reactions were carried out in a buffer identical to that used in the filter binding experiments but supplemented with 0.1 mM MgCl(2) (Mg enhances DNase I activity). To control for any effects of DNA length and the presence of Mg in the binding buffer, identical binding reactions were carried out on control DNAs generated by cleavage of plasmids carrying O(R)1-4A and O(R)1-4G with HindIII and SmaI followed by gel purification of the resulting 51-base pair operator-containing fragment and repair of the recessed HindIII end with the Klenow fragment of DNA polymerase I in the presence of [alpha-P]dATP. After a 10-min incubation of these DNAs with varying amounts of repressor at 23 °C, sufficient DNase I was added to give, on average, one cleavage/DNA molecule in 5 min of further incubation. Following incubation with the nuclease, the DNAs were extracted with phenol/chloroform, precipitated with ethanol, suspended in 90% formamide dye mix, and denatured by heating at 90 °C. The samples were then treated as described above. The dissociation constants were determined from three different autoradiograms resulting from separate experiments.


RESULTS

Effects of the Base Pairs at Position 6 of O(R)1 and O(R)3 on Repressor-Operator Affinity

We previously reported that an AbulletT GbulletC change at position 4 had a much greater effect on operator affinity for repressor in the context of O(R)1 than in the context of O(R)3(13) . Since O(R)1 and O(R)3 differ in sequence at position 6, we tested the role of the base pair at this position by swapping the base pairs between O(R)1 and O(R)3. To assess any cooperative role of the position 4 and position 6 base pairs, we made these swaps in the context of either an AbulletT or a GbulletC at position 4 of O(R)1 and O(R)3. Table 1shows that at both 50 and 100 mM KCl, changing the base pair at position 4 from an AbulletT to a GbulletC lowers the affinity of repressor for operators bearing a CbulletG at position 6 to a much greater extent than when position 6 is occupied by a TbulletA base pair. For example, in the context of O(R)1, an AbulletT GbulletC substitution at position 4 lowers the affinity of repressor for operators with a CbulletG at position by almost 200-fold at 100 mM KCl (compare lines 1 and 3) whereas, under the same conditions, when position 6 is a TbulletA, the same AbulletT GbulletC substitution at position 4 decreases the affinity of repressor by only 2.5-fold (lines 2 and 4). Similarly, in the context of O(R)3 when position 6 is a CbulletG, an AbulletT GbulletC change at position 4 decreases the affinity of repressor by 175-fold (lines 6 and 8), whereas when the base pair at position 6 is a TbulletA, the same change at position 4 actually increases the affinity of repressor by 1.5-fold (lines 5 and 7). These results demonstrate that the position 4 context effect observed for the wild-type operators is due, in large part, to the difference in sequence of these operators at position 6.



We showed previously that ionic interactions play a much greater role in stabilizing the complexes formed between repressor and the position 4 variants of O(R)1 and O(R)3 than they do in complexes formed between repressor and the wild-type operators. To determine the extent to which the identity of the base pair at position 6 affects the ionic interactions of repressor with O(R)1, O(R)3 and their position 4 variants, the dependence of K(d) on monovalent cation concentration was measured. In O(R)1, when the base at position 4 is an AbulletT, a position 6 CbulletG TbulletA change results in an operator (O(R)1-4A6T) whose affinity for repressor decreases 11-fold when the ionic strength is shifted from 50 to 100 mM KCl. This contrasts with the insensitivity of the affinity of repressor for wild-type O(R)1 (O(R)1-4A6C) to changes in ionic strength (Table 1). Conversely, when position 4 is occupied by a GbulletC base pair in O(R)1, the position 6 CbulletG TbulletA change results in an operator (O(R)1-4G6T) whose affinity for repressor is only moderately decreased (2.5-fold) by increasing the ionic strength from 50 to 100 mM KCl, while under identical conditions the affinity of repressor for O(R)1-4G6C decreases by 18.5-fold. Similar sequence-dependent effects are observed for the position 4 and 6 variants of O(R)3. When the base pair at position 4 of O(R)3 is a GbulletC, changing the base pair at position 6 from a TbulletA to a CbulletG increases the ionic strength dependence of the repressor-operator complex from one that is essentially independent of salt concentration to one that is destabilized 11-fold as the salt concentration increases from 50 to 100 mM KCl. As seen in the context of O(R)1, changing position 6 has the opposite effect on ionic strength dependence of affinity when position 4 is first mutated; when position 4 in O(R)3 is an AbulletT, changing the base pair at position 6 from a TbulletA to a CbulletG decreases the ionic strength sensitivity of repressor's affinity. Together, the data from the above analyses show that neither the base pair at position 4 nor the base pair at position 6 specifies the ionic strength dependence of the affinity of repressor for these operators. Instead, a specific combination of position 4 and position 6 base pairs determines the ionic strength dependence of the affinity of repressor.

The differing ionic strength dependences of complexes formed between repressor and the position 6 mutants of the wild-type and position 4 variant O(R)1 and O(R)3 indicate that the position 6 base pair has a major role in determining the degree that ionic interactions contribute to operator strength. Differences in the ionic strength dependences of a protein-DNA complex can be due to changes in the strength and/or number of ionic interactions between protein and DNA. Since all 434 repressor-operator complexes studied thus far appear to have an identical number of contacts with the DNA phosphate backbone (5, 6, 21, 22, 23) , the observed differences in ionic contributions to binding energy must be due to an alteration in the strength of ionic interactions. This observation implies that the structures of these repressor-operator complexes differ and that these structural differences are mediated, at least in part, by cooperative interaction between positions 4 and 6 base sequences.

The Role of a Position 6 Inclusive Oligo-dAbulletOligo-dT Tract in Repressor's Affinity for Operator

Both the magnitude of repressor's position 4 preference and the ionic strength sensitivity of its affinity are dependent upon the identity of the base pair at position 6. Since the data in Table 1suggest that the structures of the repressor-operator complexes vary with position 6 base pair, we wished to examine how position 6 exerts its effect. X-ray crystallographic and chemical interference data suggest that the major and minor groove surfaces of the base pair at position 6 in O(R)1 and O(R)3 are not contacted by repressor(5, 6) . (^2)This is consistent with the suggestion that the influence of the base pair at position 6 on operator strength involves some sequence-dependent structural aspect of the DNA. The TbulletA base pair at position 6 in O(R)3 is part of a 5-base oligo-dAbulletoligo-dT tract that dominates its central sequence (see Fig. 1). Swapping the base pair at position 6 between O(R)1 and O(R)3 shortens the length of this continuous tract to 3 base pairs in O(R)3, while increasing the length of this tract in O(R)1 from 3 to 4 (Fig. 1). Oligo-dAbulletoligo-dT tracts greater than or equal to 4 base pairs are known to introduce an intrinsic bend in DNA(24) . The direction of the bend predicted to be produced by the central sequence of O(R)3 would be in phase with the bend induced by 434 repressor upon binding(6, 24) . Since operators whose structure more closely resembles that of the final complex bind repressor with higher affinity(25, 26) , it is reasonable to hypothesize that the influence of the base pair at position 6 on the affinity of repressor for position 4 variants of O(R)1 and O(R)3 results from its participation in an oligo-dAbulletoligo-dT sequence-induced bend. If such sequence-induced bending suppresses repressor's apparent position 4 recognition, then the presence of any base pair other than a TbulletA at position 6 should obviate those effects.

Fig. 2presents data comparing the affinity of repressor at 50 and 100 mM KCl for operators with either GbulletC, AbulletT, TbulletA, or CbulletG base pairs at position 6 in the context of either an AbulletT or a GbulletC base pair at position 4 in both O(R)1 and O(R)3. In the context of an AbulletT base pair at position 4 (Fig. 2, A and C) a TbulletA at position 6 is not the only base pair that results in an operator whose affinity for repressor is significantly dependent upon ionic strength. In the context of a position 4 AbulletT, only a position 6 CbulletG markedly reduces the ionic strength dependence of the affinity of repressor for these operators. Likewise, in operators with a GbulletC base pair at position 4 (Fig. 2, B and D), a position 6 TbulletA is not the only base pair that results in an operator whose affinity for repressor is only weakly dependent upon ionic strength. In O(R)1-4G, both a GbulletC and a TbulletA base pair permit repressor to bind in a fashion that is relatively independent of ionic strength. Similarly in O(R)3-4G, only a position 6 CbulletG base pair generates an operator whose affinity for repressor is markedly dependent upon ionic strength. Consideration of the data in Fig. 2also shows that a position 6 TbulletA is not the only base pair that can suppress the deleterious effect of an AbulletT GbulletC change at position 4 on the affinity of repressor for operator. In the context of O(R)3, at both 50 and 100 mM KCl, changing position 6 from a TbulletA to a GbulletC or an AbulletT also results in an operator whose affinity for repressor is relatively unaffected by an AbulletT GbulletC change at position 4 (compare Fig. 2, C and D). In this sequence context, only a position 6 CbulletG base pair results in an operator whose affinity for repressor is strongly decreased by position 4 substitution. Comparing Fig. 2, A and B, reveals a qualitatively similar but not quantitatively identical pattern of position 6 effects on discrimination at position 4 in O(R)1. Taken together, these data do not support a model where the participation of the base pair at position 6 in an oligo-dAbulletoligo-dT tract defines its role in determining either the affinity of repressor or the ionic strength sensitivity of the complexes between repressor and position 4 variants of O(R)1 and O(R)3.


Figure 2: Effect of position 6 substitution on the affinity of 434 repressor for O(R)1, O(R)3 and their position 4 variants. Plotted is the K versus the base at position 6 in O(R)1 (A), O(R)1-4G (B), O(R)3-4A (C), and O(R)3 (D). Values of the dissociation constants measured at 50 (hatched bars) and 100 (filled bars) mM KCl are shown. Note scale difference in panelsA and C from B and D



Effect of the Base Pair at Position 5 on the Affinity of 434 Repressor for O(R)1 and O(R)3

Comparison of the data plotted in Fig. 2, A and B, with that in C and D reveals that the degree to which ionic strength affects the affinity of repressor for position 6 variants is not the same for O(R)1 and O(R)3 even when the base pair at position 4 is the same. Moreover, as discussed above, changing the base pair at position 6 does not have quantitatively identical effects on the affinity of repressor for the position 4 variants of O(R)1 and O(R)3. These data suggest the different base pairs at position 5 in O(R)1 and O(R)3 must also have a role in determining the affinity of repressor for its operators. The affinities of repressor for position 4-6 variants of O(R)1 and O(R)3 are displayed in Table 2. By comparing the affinity of repressor for operators that differ in sequence only at position 5, we can determine the effect of position 5 on operator affinity for repressor.



In O(R)1, position 5 is occupied by an AbulletT base pair, and in O(R)3 position 5 is a TbulletA. At 100 mM KCl, changing the base pair at position 5 of wild-type O(R)1 from an AbulletT to a TbulletA decreases the affinity of repressor for the resulting operator by 2-fold (Table 2, compare lines 1 and 5). Conversely, in O(R)3, the same position 5 AbulletT TbulletA change increases the affinity of repressor for the resulting operator by 4-fold (Table 2, compare lines 12 and 16). Thus, the net result of swapping the base pairs at position 5 between wild-type O(R)1 and wild-type O(R)3 is to increase the degree to which repressor discriminates between these operators by 2-fold.

By comparing the ionic strength dependence of the affinity of repressor for two operators with the same position 4 and position 6 base, we can deduce the degree to which a TbulletA versus an AbulletT base pair at position 5 contributes to the affinity of repressor for each operator and the ionic strength dependence of repressor-operator complexes. Similarly by comparing the effect of an AbulletT GbulletC change at position 4 in the context of identical base pairs at position 6, we can determine the contribution of the base pair at position 5 to position 4 preference. Sequential comparisons of this type reveal the context-dependent nature of the effect of a base pair substitution at position 5.

When the base pair at position 4 is an AbulletT, in every position 6 context except one (6T), the presence of a TbulletA base pair at position 5 results in a binding site whose affinity for repressor is more ionic strength dependent than when position 5 is an AbulletT (Table 2, compare lines 1-4 with 5-8). Conversely, when the base pair at position 4 is a GbulletC, in every position 6 context, a position 5 AbulletT TbulletA substitution results in an operator whose affinity for repressor is less dependent on ionic strength (Table 2, compare lines 9-12 with 13-16). Furthermore, in the context of a position 4 AbulletT, a position 5 AbulletT TbulletA substitution consistently alters the ionic strength dependence of the affinity of repressor for operator by only 1.5-fold (Table 2, compare lines 1-4 with 5-8). The same substitution lowers the ionic strength dependence of the affinity of repressor for operators with a position 4 GbulletC by as little as 1.5-fold (Table 2, compare line 9 with 13) to as much as 45-fold (Table 2, compare line 11 with 15). Taken together, these results demonstrate that the degree to which the affinity of repressor for position 4 variants is altered by ionic strength is completely dependent upon the identity of the base pairs at positions 5 and 6. Moreover, the effects of the base pairs at positions 4-6 are not independent of each other; specific combinations of base pairs at these three positions cooperate to generate different ionic strength dependence phenotypes.

Although the magnitude of the effect of a position 4 AbulletT GbulletC change differs depending on the base pair at position 6 (for example see Table 1), in every position 6 context the identity of the base pair at position 5 only slightly influences the magnitude of the affinity change resulting from this mutation at position 4. For example, at high salt, when the base pair at position 6 is a CbulletG, changing the base pair at position 4 from an AbulletT to a GbulletC lowers repressors affinity by 175-fold in the context of an TbulletA base pair at position 5 (Table 2, compare lines 5 and 13). Similarly, the same position 4 change has a 195-fold effect on affinity in the context of a position 5 AbulletT base pair (Table 2, compare lines 1 and 9). While changing position 5 does not greatly affect the magnitude of repressor's position 4 preference, changes at position 5 do influence repressor's position 6 preference. For example, in an operator bearing a position 4 GbulletC, at high salt, repressor prefers a TbulletA base pair at position 6 over an AbulletT by 10-fold in the context of an AbulletT at position 5 (Table 2, compare lines 11 and 12), while when position 5 is occupied by a TbulletA base pair, repressor now shows a 3-fold preference for an AbulletT over a TbulletA base pair at position 6 (lines 15 and 16). Thus, mutating position 5 can change repressor's position 6 preference by 30-fold. A 15-fold change in position 6 preference is mediated by altering position 5 is the context of a position 4 GbulletC ( Table 2compare lines 10 and 11 with 14 and 15). In addition to these position 5-dependent reversals in the affinity order of repressor for position 6, swapping a TbulletA for an AbulletT at position 5 can also produce more subtle effects on the magnitude of repressor's position 6 preference; for example, compare 4G5T6CT with 4G5A6CT ( Table 2compare lines 13 and 16 with 9 and 12). Taken together, these data show that position 5 has a much greater effect on repressor's discrimination at position 6 than it does on discrimination at position 4.

Dependence of the Position 4, 5, and 6 Preferences of 434 Repressor on the Identity of the Amino Acid at Position 33 of Repressor

Glutamine 33 of repressor makes both hydrogen bond and van der Waals' contacts with the thymine at position 4` of O(R)1(5, 8) . Changing the base pair at position 4 from an AbulletT to a GbulletC likely eliminates both of these contacts(6) . We wished to determine whether the contribution of the base pair at position 4 to the ionic strength sensitivity of repressor's affinity for its operators and its position 5 and 6 preferences are mediated by glutamine 33. Previous work showed that substitution of glutamine 33 with an alanine reduces the degree to which repressor discriminates between operators whose sequence differs at position 4(8, 14) . This reduction indicates a loss or alteration of the contact between the amino acid at position 33 and that base pair. We reasoned that if the link between the base pair at position 4 and the base pairs at positions 5 and 6 is the contact between glutamine 33 and the base at position 4`, then substitution of glutamine 33 with an alanine should reduce the specificity at position 4 and therefore result in complexes where the ionic strength sensitivity of the dissociation constants is independent of the base at position 4. Table 3shows that, in agreement with previous results(8, 14) , changing the amino acid at position 33 of repressor to an alanine creates a protein whose position 4 specificity is markedly reduced. Moreover, the affinity of this protein for O(R)1 and O(R)3 is independent of ionic strength regardless of the base pair at position 4. This implies that the position 4 contribution to the ionic strength dependence of wild-type repressor binding involves contact by glutamine 33.



Dependence of 434 Repressor-Operator Affinity and Ionic Strength Sensitivity on a Contact with the Base at Operator Position 4`

The above experiment does not distinguish whether the hydrogen bond, the van der Waals' contact or both couples the position 4 base pair to the effect of the base pairs at positions 5 and 6. To distinguish among these possibilities, the affinity and ionic strength sensitivity of the binding of wild-type repressor to an O(R)1-4G operator bearing a C at position 4` was determined. As mentioned above, glutamine 33 of repressor makes a van der Waals' contact with the 5-methyl group of the thymine at position 4` of O(R)1(5) . In O(R)1-4G, repressor presumably can not make this van der Waals' contact because the corresponding base at position 4` is a cytosine and therefore lacks the 5-methyl group present on a thymine. Replacing the position 4` C in O(R)1-4G with a C repressor should reestablish a contact with this base. If the contribution of the base pair at position 4 to the affinity and ionic strength sensitivity of the binding of repressor relies on its participation in this contact, then reestablishment of this interaction in O(R)1-4G should result in an operator that binds more tightly than the unsubstituted operator and whose affinity for repressor is no longer strongly dependent on ionic strength. The data in Table 4show that repressor's affinity for O(R)1-4GbulletC is decreased 6-fold by an increase in KCl concentration from 50 to 100 mM. This effect is smaller than that observed in the experiments summarized in Table 1, presumably because of the presence of MgCl(2) in these experiments(27) . Under the conditions of this experiment, the affinity of repressor for O(R)1-4AbulletT is reduced by 1.5-fold. Consistent with reestablishment of a favorable contact between repressor and O(R)1-4GbulletC, the affinity of repressor for this operator at 50 mM KCl is approximately 5-fold higher than its affinity for O(R)1-4GbulletC. Moreover, the results of affinity measurements at 50 and 100 mM KCl demonstrate that the ionic strength dependence of the affinity of wild-type repressor for O(R)1-4GbulletC is identical to the ionic strength dependence of its affinity for O(R)1-4AbulletT. These results are consistent with the hypothesis that reestablishment of the contact between the base at position 4` and glutamine 33 is sufficient to reduce the ionic strength dependence of the affinity of repressor for O(R)1-4G. Thus, the status of the contact at position 4` plays a pivotal role in determining the ionic strength sensitivity as well as the affinity of repressor for these operators.




DISCUSSION

Previous work has shown that 434 repressor distinguishes between O(R)1 and O(R)3 in large measure because of the sequence difference at position 4 of these operators(8, 13, 14) . The results presented here show that the affinity of repressor for O(R)1 and O(R)3 and two of their position 4 variants is strongly influenced by the base pairs at positions 5 and 6. Moreover, the degree to which position 4 substitutions affect operator strength is tempered by the identity of the base pairs at positions 5 and 6. We have shown here that these base pairs influence the affinity of repressor by modulating the degree to which ionic interactions contribute to the stability of the repressor-operator complex.

The influence of the base pair at position 6 on the affinity of repressor for an operator is exemplified by the observed effects of base pair substitutions at that position on the affinity of repressor at high salt concentration. The base pair at position 6 that results in lower or higher repressor-operator affinity depends upon which base pair is at position 4 (Table 1). Moreover, though 434 repressor discriminates between wild-type O(R)1 and O(R)3 by 5-fold throughout the salt concentration range studied ( Table 1and (13) ), our results show that a position 6 swap between O(R)1 and O(R)3 generates operators whose affinity for repressor now differs by 45-fold in this range of salt concentration. Furthermore, if O(R)1 and O(R)3 were to have both a CbulletG base pair at position 6, then repressor would discriminate between them by 40-fold at 50 mM KCl and 350-fold at 100 mM KCl. These data clearly demonstrate that the identity of the base pair at position 6 of O(R)1 and O(R)3 plays an important role in determining the degree to which repressor discriminates between these operators. The identity of the base pair at position 5 of O(R)1 and O(R)3 plays a similar, though less dramatic, role in determining the degree to which repressor discriminates between these operators. The net result is that swapping the base pairs at position 5 between wild-type O(R)1 and wild-type O(R)3 increases the degree to which repressor discriminates between these operators by 2-fold (Table 2). Changes at position 5 have a much larger effect on repressor's position 6 preference. Moreover, since the base pair at position 6 alters or even reverses repressor's position 4 preference and the base pair at position 5 modulates position 6 preference, position 5 indirectly influences repressor's position 4 preference. This influence is only apparent in certain position 6 contexts. Therefore, the base pairs at positions 5 and 6 combine to influence repressor's preference at position 4.

Since neither the major nor minor groove surface of the base pairs at positions 5 and 6 in O(R)1 and O(R)3 are contacted by repressor(6, 10) , we suggest that the base pairs at positions 5 and 6 modulate repressor's affinity for operator by changing operator structure in the complex. Dissimilar salt dependences are thought to be the result of differences in the number and/or strength of charged interactions between protein and the DNA phosphate backbone(28) . Two lines of evidence support the idea that differences in the ionic strength sensitivity of repressor's affinity for position 4 variants of O(R)1 and O(R)3 result from alterations in the strength of a shared number of ionic contacts rather than a difference in the actual number of contacts. The number of protein-phosphate backbone contacts identified by ethylnitrosourea interference experiments is identical for all repressor complexes tested(21, 22) . In addition, comparison of the crystal structures of amino acids 1-69 in complex with O(R)1, O(R)3, and O(R)1-4G reveals an identical number of direct interactions between the protein and the phosphate backbone of these operators(5, 6) . (^3)Assuming that all repressor-operator complexes studied here have the same number of ionic interactions, an increase in the ionic strength sensitivity of a particular repressor-operator complex, as a result of changes in operator sequence, indicates that the change in operator sequence caused a reduction in the strength of ionic interactions within that complex. In this view, the base pairs at positions 5 and 6 modulate the affinity of repressor for position 4 variants of O(R)1 and O(R)3 by altering the contribution of ionic interactions to the overall binding energy. Our data also show that the combination of base pairs at positions 5 and 6 that result in a lower or higher dependence of repressor-operator affinity on ionic strength depends upon which base pair is at position 4.

The simplest interpretation of these results, in light of this structural model, is that the sequence-dependent structural alterations conferred by a particular combination of bases at positions 5 and 6 that generate a favorable configuration of the phosphates when position 4 is an AbulletT must result in a less favorable configuration when position 4 is a GbulletC. In agreement with this, differences in the sensitivity of a repressor-operator complex to cleavage by hydroxyl radical depend upon the position 5 and 6 context of the position 4 base (13) . We show here that these dissimilar ionic strength sensitivities are a result of the base pair composition of O(R)1 and O(R)3 at positions 5 and 6.

An alternative explanation of the ionic strength and affinity data suggests that instead of reflecting changes the protein-DNA interface, the effect of position 4, 5, and 6 changes on the strength of repressor-operator complexes may indicate alterations in the conformation of repressor's dimer interface. Support for this idea comes from the observation that mutagenizing amino acids involved in forming the amino-terminal dimer interface can alter repressor's central sequence preferences(11) .^2 The data presented here can neither prove nor dismiss this alternative. However, the observed differences in ionic strength sensitivity arise from base pair changes near the locations of known protein-DNA phosphate backbone interactions (10) . Moreover, these differences in ionic strength sensitivity are correlated with changes in the structure of the DNA within these operators(13) . These observations suggest that changes in operator sequence at positions 4-6 alter the interactions between the repressor and the operator DNA phosphate backbone.

The observation that a Gln Ala mutation allows repressor to form complexes with O(R)1-4G and O(R)3-4A that are relatively insensitive to salt concentration implies that the strong ionic strength dependence of O(R)1-4G- and O(R)3-4A-wild-type repressor complexes is due to altered contacts between glutamine 33 and the position 4 base. The precise nature of this structural alteration can not be determined from our data. Our data do suggest, however, that simply losing a van der Waals' contact with the base at position 4` is not sufficient to cause the stability of a repressor-operator complex to become sensitive to ionic strength. For example, the affinities of repressor for wild-type O(R)3 or O(R)1-4G-6T are not greatly affected by changes in salt concentration, even though no van der Waals' contact between Gln and the base at position 4` appears possible. Why then are some of wild-type repressor-operator complexes affected by ionic strength, but none of the Ala-repressor-operator complexes are? We suggest that Ala may make some residual contact with position 4 in all operators, and it is this contact that prevents ionic strength from affecting complex formation. Support for the suggestion that Ala contacts the base at position 4 comes from measurements of the affinity of Ala for a set of symmetric reference operators in which the base pair at position 4 of each half-site (positions 4 and 11` in Fig. 1) is varied(8) . These results demonstrate that Ala is capable of strongly distinguishing between AbulletT, AbulletU, GbulletC, and GbulletC base pairs at position 4 of these operators. This observation implies that, in some way, the base pair at position 4 is recognized by the Ala protein.

The idea that some form(s) of contact between the position 4 base and amino acid 33 can reduce the ionic strength sensitivity of a repressor-operator complex suggests a possible explanation for the observed effects of position 5/6 base pair substitutions. That is, only particular position 5/6 base pair combinations permit repressor's amino acid 33 to make a contact with the position 4 base pair, thereby rendering the stability of the complex relatively impervious to changes in salt concentration, while other position 5/6 combinations do not. Alternatively, the status of the contact at position 4 could be solely determined by the identity of the base pair at that position, while the overall affinity and ionic strength sensitivity of the resulting complex is modulated by the response of the repressor to the position 5/6 sequence context in which that contact is (or is not) made. The results presented here neither support nor exclude either mechanism.

The idea that the contact at position 4 has a role in establishing the ionic strength sensitivity of repressor's affinity is directly supported by the observation that replacing the position 4` C in O(R)1-4G with a C both increases operator affinity for repressor and decreases the ionic strength dependence of the affinity of repressor for O(R)1-4G. The independence of repressor's affinity for O(R)1-4GbulletC from salt concentration demonstrates that reestablishment of a contact involving the 5-methyl group at position 4` is sufficient to eliminate the strong ionic strength sensitivity observed for the affinity of repressor for unmodified O(R)1-4G (Table 4). Furthermore, the fact that the ionic strength sensitivities of the repressor-O(R)1-4AbulletT and repressor-O(R)1-4GbulletC complexes are indistinguishable suggests that the presence of a 5-methyl group at operator position 4` is sufficient to generate ionic strength insensitivity in the context of an O(R)1 central sequence.

Taken together, our results imply that all of the bases that differ between O(R)1 and O(R)3, positions 4, 5 and 6, are important in determining repressor's affinity. A difference in base sequence at position 4 is relevant because it determines, at least, the presence or absence of a direct contact with repressor. Positions 5 and 6 are relevant because their identity modulates the ionic contribution to the binding energy. Moreover, there is an intimate connection between the status of the contact at position 4 and the nature of the influence of the base pairs at positions 5 and 6 on the affinity of repressor. The sequence of the noncontacted bases at operator positions 5 and 6 can modify preference of 434 repressor for a base at a contacted position. As suggested by the ionic strength dependence data shown here and our earlier results(15) , the effect of these noncontacted bases is presumably through alteration of the DNA structure in the repressor-operator complex.

Having established that positions 5 and 6 modulate repressor's position 4 preference in a manner that depends on ionic strength leads to the question of why the phage evolved the particular combination of bases found at positions 4-6 of O(R)1 and O(R)3. One possible answer to this question emerges from consideration of our data in light of recent measurements which establish that E. coli's cytoplasm contains 140 mM K(29) . Our results suggest that at this salt concentration, changes in the base sequence at positions 4, 5, or 6 of O(R)1 or O(R)3 would result in a greater separation of repressor's affinity for these operators. Given that the genetic switch governing 434 phage development requires maintaining a small difference in the affinity of repressor for O(R)1 and O(R)3(1, 2) , it is striking that by making use of sequence-dependent modulations of both ionic and nonionic interactions in the repressor-operator complex the phage has evolved a pair of operators that fulfill this requirement.


FOOTNOTES

*
This work supported by National Institutes of Health Grant GM42138. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Laboratory of Molecular Biology, NIH/NIDDK, Bldg. 5, Rm. 208, Bethesda, MD 20892-0540.

To whom correspondence should be addressed: Dept of Biological Sciences, State University of New York at Buffalo, Cooke Hall, North Campus Box 601300, Buffalo, NY 14260. Tel.: 716-645-3489; Fax: 716-645-2975; CAMGBK{at}UBVMS.CC.BUFFALO.EDU.

(^1)
The abbreviation used is: C, 5-methylcytosine.

(^2)
G. Koudelka, unpublished results.

(^3)
S. Harrison, personal communication.


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