Recognition of DNA structure by 434 repressor.

In complexes of bacteriophage 434 binding sites with 434 repressor the central 4 bp of the 14 bp site are not contacted by the protein, although changes in these bases alter binding site affinity for the repressor. Our previous data suggested that the ability of the non-contacted central bases to be overtwisted in repressor-DNA complexes governs affinity of the binding site for 434 repressor. This idea was tested by examining the affinity of two central sequence variant 434 binding sites for 434 repressor as a function of binding site average twist. The 434 repressor preferred the relatively overwound binding site to the two more underwound forms. The greatest affinity enhancement resulting from increasing twist was observed with a binding site that is relatively underwound and more resistant to twisting deformation. Consistent with the idea that 434 repressor overtwists its binding site upon DNA binding, we show that 434 repressor is capable of binding to sites bearing a single base insertion in their center (a 15mer), but binds poorly to binding sites bearing central base deletions (12mer and 13mer). The N-terminal dimer interface plays a large role in determining 434 repressor central base preferences. Mutations in this interface eliminate central base discrimination and/or site size preferences. These mutations also lead to changes in the size of the repressor footprint on the various sized DNA sites that are consistent with their binding characteristics.     

Interaction of the Mnt repressor with 37-base pair synthetic operator DNA fragments. Importance of symmetric GC pairs

Mnt repressor is indirectly responsible for the maintenance of lysogeny of the phage P22. This repressor interacts with a 21-base pair operator DNA constituting within it a 17-base pair perfect 2-fold symmetric sequence whose bases make a direct contact with the protein. We have synthesized six 37-base pair DNAs consisting of 21 base pair natural operator and its modifications in which certain symmetrically situated GC base pairs were replaced systematically with ATs to understand their importance. The binding interaction studies of Mnt repressor to such natural and modified operator DNAs reported here indicate that the GCs close to the center of symmetry make major contacts with the protein whereas, GCs nearer to the periphery form weak contacts. Methylation protection experiments indicated that when the GCs near the center of symmetry were replaced with AT, the central GC became more accessible for dimethyl sulfate methylation with possible conformational change in DNA. The circular dichroism studies indicated that upon repressor binding conformational changes in DNA takes place with a possible increase in helicity of the repressor protein.     

Non-contacted bases affect the affinity of synthetic P22 operators for P22 repressor.

The affinity of synthetic P22 operators for P22 repressor varies with the base sequence at the operator's center. At 100 mM KCl, the affinity of these operators for P22 repressor varies over a 10-fold range. Dimethylsulfate protection experiments indicate that the central bases of the P22 operator are not contacted by the repressor. The KD for the complex of P22 repressor with an operator bearing central T-A bases (9T) increases less than 2-fold between 50 and 200 mM KCl, whereas the KD for the complex of repressor with an operator bearing central C-G bases (9C) increases 10-fold in the same salt range. The DNase I cleavage patterns of both bound and unbound P22 operators also vary with central base sequence. The DNase I pattern of the repressor-9C operator complex changes markedly with salt concentration, whereas that of the 9T operator-repressor complex does not. These changes in nuclease digestion pattern thereby mirror the salt-dependent changes in the P22 operator's affinity for repressor. P22 repressor protects the central base pair of the 9T operator from cleavage by the intercalative cleavage reagent Cu(I)-phenanthroline, while repressor does not protect the central bases of the 9C operator. Together these data indicate that central base pairs affect P22 operator strength by altering the structure of the unbound operator and the repressor-operator complex.     

Bending of synthetic bacteriophage 434 operators by bacteriophage 434 proteins.

The extent of DNA bending induced by 434 repressor, its amino terminal DNA binding domain (R1-69), and 434 Cro was studied by gel shift assay. The results show that 434 repressor and R1-69 bend DNA to the same extent. 434 Cro-induced DNA bends are similar to those seen with the 434 repressor proteins. On approximately 265 base pair fragments, the cyclic AMP receptor protein of Escherichia coli (CRP) produces larger mobility shifts than does 434 repressor. This indicates that the 434 proteins bend DNA to a much smaller extent than does CRP. The effects of central operator sequence on intrinsic and 434 protein-induced DNA bending was also examined by gel shift assay. Two 434 operators having different central sequences and affinities for 434 proteins display no static bending. The amount of gel shift induced by 434 repressor on these operators is identical, showing that the 434 repressor bends operators with different central sequences to the same extent. Hence, mutations in the central region of the operator do not influence the bent structure of the unbound or bound operator.     

Selection and design of high affinity DNA ligands for mutant single-chain derivatives of the bacteriophage 434 repressor

Single-chain repressor RRTRES is a derivative of bacteriophage 434 repressor, which contains covalently dimerized DNA-binding domains (amino acids 1-69) of the phage 434 repressor. In this single-chain molecule, the wild type domain R is connected to the mutant domain RTRES by a recombinant linker in a head-to-tail arrangement. The DNA-contacting amino acids of RTRES at the -1, 1, 2, and 5 positions of the a3 helix are T, R, E, S respectively. By using a randomized DNA pool containing the central sequence -CATACAAGAAAGNNNNNNTTT-, a cyclic, in vitro DNA-binding site selection was performed. The selected population was cloned and the individual members were characterized by determining their binding affinities to RRTRES. The results showed that the optimal operators contained the TTAC or TTCC sequences in the underlined positions as above, and that the Kd values were in the 1×10-12 mol/L-1×10-11mol/L concentration range. Since the affinity of the natural 434 repressor to its natural operator sites is in the 1×10-9 mol/L range, the observed binding affinity increase is remarkable. It was also found that binding affinity was strongly affected by the flanking bases of the optimal tetramer binding sites, especially by the base at the 5′ position. We constructed a new homodimeric single-chain repressor RTRESRTRES and its DNA-binding specificity was tested by using a series of new operators designed according to the recog-nition properties previously determined for the RTRES domain. These operators containing the con-sensus sequence GTAAGAAARNTTACN or GGAAGAAARNTTCCN (R is A or G) were recognized by RTRESRTRES specifically, and with high binding affinity. Thus, by using a combination of random selection and rational design principles, we have discovered novel, high affinity protein-DNA inter-actions with new specificity. This method can potentially be used to obtain new binding specificity for other DNA-binding proteins.     

Monovalent cations regulate DNA sequence recognition by 434 repressor

The bacteriophage 434 repressor distinguishes between its six naturally occurring binding sites using indirect readout. In indirect readout, sequence-dependent differences in the structure and flexibility of non-contacted bases in a protein's DNA-binding site modulate the affinity of DNA for protein. The conformation and flexibility of a DNA sequence can be influenced by the interaction of the DNA bases or backbone with solution components. We examined the effect of changing the cation-type present in solution on the stability and structure of 434 repressor complexes with wild-type and mutant OR1 and OR3, binding sites that differ in their contacted and non-contacted base sequences. We find that the affinity of repressor for OR1, but not for OR3, depends remarkably on the type and concentration of monovalent cation. Moreover, the formation of a stable, specific repressor-OR1 complex requires the presence of monovalent cations; however, repressor-OR3 complex formation has no such requirement. Changing monovalent cation type alters the ability of repressor to protect OR1, but not OR3, from *OH radical cleavage. Altering the relative length of the poly(dA) x poly(dT) tract in the non-contacted regions of the OR1 and OR3 can reverse the cation sensitivity of repressor's affinities for these two sites. Taken together these findings show that cation-dependent alterations in DNA structure underlies indirect readout of DNA sequence by 434 repressor and perhaps other proteins.     

Indirect Readout of DNA Sequence by P22 Repressor: Roles of DNA and Protein Functional Groups in Modulating DNA Conformation

The repressor of bacteriophage P22 (P22R) discriminates between its various DNA binding sites by sensing the identity of non-contacted base pairs at the center of its binding site. The “indirect readout” of these non-contacted bases is apparently based on DNA's sequence-dependent conformational preferences. The structures of P22R–DNA complexes indicate that the non-contacted base pairs at the center of the binding site are in the B′ state. This finding suggests that indirect readout and therefore binding site discrimination depend on P22R's ability to either sense and/or impose the B′ state on the non-contacted bases of its binding sites. We show here that the affinity of binding sites for P22R depends on the tendency of the central bases to assume the B′-DNA state. Furthermore, we identify functional groups in the minor groove of the non-contacted bases as the essential modulators of indirect readout by P22R. In P22R–DNA complexes, the negatively charged E44 and E48 residues are provocatively positioned near the negatively charged DNA phosphates of the non-contacted nucleotides. The close proximity of the negatively charged groups on protein and DNA suggests that electrostatics may play a key role in the indirect readout process. Changing either of two negatively charged residues to uncharged residues eliminates the ability of P22R to impose structural changes on DNA and to recognize non-contacted base sequence. These findings suggest that these negatively charged amino acids function to force the P22R-bound DNA into the B′ state and therefore play a key role in indirect readout by P22R.     

Orientation of the putative recognition helix in the DNA-binding domain of Hin recombinase complexed with the hix site

On the basis of sequence similarity with other known DNA-binding proteins, the DNA-binding domain of Hin recombinase, residues 139-190, is thought to bind DNA by a helix-turn-helix motif. Two models can be considered that differ in the orientation of the recognition helix in the major groove of DNA. One is based on the orientation of the recognition helix found in the 434 repressor (1-69) and lambda repressor-DNA cocrystals, and the other is based on the NMR studies of lac repressor headpiece. Cleavage by EDTA.Fe attached to a lysine side chain (Ser183----Lys183) near the COOH terminus of Hin(139-184) reveals that the putative recognition helix is oriented toward the center of the inverted repeats in a manner similar to that seen in the 434 and lambda repressor-DNA cocrystals.     

Determinants of bacteriophage 933W repressor DNA binding specificity

We reported previously that 933W repressor apparently does not cooperatively bind to adjacent sites on DNA and that the relative affinities of 933W repressor for its operators differ significantly from that of any other lambdoid bacteriophage. These findings indicate that the operational details of the lysis-lysogeny switch of bacteriophage 933W are unique among lambdoid bacteriophages. Since the functioning of the lysis-lysogeny switch in 933W bacteriophage uniquely and solely depends on the order of preference of 933W repressor for its operators, we examined the details of how 933W repressor recognizes its DNA sites. To identify the specificity determinants, we first created a molecular model of the 933W repressor-DNA complex and tested the predicted protein-DNA interactions. These results of these studies provide a picture of how 933W repressor recognizes its DNA sites. We also show that, opposite of what is normally observed for lambdoid phages, 933W operator sequences have evolved in such a way that the presence of the most commonly found base sequences at particular operator positions serves to decrease, rather than increase, the affinity of the protein for the site. This finding cautions against assuming that a consensus sequence derived from sequence analysis defines the optimal, highest affinity DNA binding site for a protein.     

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

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

Sequence requirement for specific interaction of an enhancer binding protein (EBP1) with DNA.

Short DNA sequence motifs have been identified in viral and cellular enhancers which represent the binding sites for a variety of trans- acting factors. One such HeLa cell factor, EBP1, has been purified and shown to bind to sequences in the SV40 enhancer. The PRDII element in the human beta-interferon gene regulatory element (IRE) shows strong sequence similarity to the EBP1 binding site in the SV40 enhancer. We demonstrate here that EBP1 binds to its sites in the SV40 enhancer and IRE in a similar manner, making base specific contacts over one complete turn of the DNA double helix. Mutational analysis of the EBP1 sites in the IRE and SV40 enhancer has identified the DNA sequence requirements necessary for specific EBP1/DNA complex formation. In addition, 34 DNA sequences related to the EBP1 binding site were analysed for their ability to bind EBP1. Sequences constituting high affinity binding sites possess the sequence 5'-GG(N)6CC-3'. Single base pair changes in the region between the conserved Gs and Cs can generally be tolerated although it is clear that these intervening bases contribute to binding affinity. Mutations in the recognition site which could lead to gross structural changes in the DNA abolish EBP1 binding.     

The P1 phage replication protein RepA contacts an otherwise inaccessible thymine N3 proton by DNA distortion or base flipping

The RepA protein from bacteriophage P1 binds DNA to initiate replication. RepA covers one face of the DNA and the binding site has a completely conserved T that directly faces RepA from the minor groove at position +7. Although all four bases can be distinguished through contacts in the major groove of B-form DNA, contacts in the minor groove cannot easily distinguish between A and T bases. Therefore the 100% conservation at this position cannot be accounted for by direct contacts approaching into the minor groove of B-form DNA. RepA binding sites with modified base pairs at position +7 were used to investigate contacts with RepA. The data show that RepA contacts the N3 proton of T at position +7 and that the T=A hydrogen bonds are already broken in the DNA before RepA binds. To accommodate the N3 proton contact the T₊₇ /A_+7^′ base pair must be distorted. One possibility is that T₊₇ is flipped out of the helix. The energetics of the contact allows RepA to distinguish between all four bases, accounting for the observed high sequence conservation. After protein binding, base pair distortion or base flipping could initiate DNA melting as the second step in DNA replication.     

DNA sequence determinants for binding of the Escherichia coli catabolite gene activator protein.

The consensus DNA site for binding of the Escherichia coli catabolite gene activator protein (CAP) is 22 base pairs in length and is 2-fold symmetric: 5'-AAATGTGATCTAGATCACATTT-3'. Positions 4 to 8 of each half of the consensus DNA half-site are the most strongly conserved. In this report, we analyze the effects of substitution of DNA base pairs at positions 4 to 8, the effects of substitution of thymine by uracil and by 5-methylcytosine at positions 4, 6, and 8, and the effect of dam methylation of the 5'-GATC-3' sequence at positions 7 to 10. All DNA sites having substitutions of DNA base pairs at positions 4 to 8 exhibit lower affinities for CAP than does the consensus DNA site, consistent with the proposal that the consensus DNA site is the ideal DNA site for CAP. Specificity for T:A at position 4 appears to be determined solely by the thymine 5-methyl group. Specificity for T:A at position 6 and specificity for A:T at position 8 appear to be determined in part, but not solely, by the thymine 5-methyl group. dam methylation has little effect on CAP.DNA complex formation. The thermodynamically defined consensus DNA site spans 28 base pairs. All, or nearly all, DNA determinants required for maximal affinity for CAP and for maximal thermodynamically defined CAP.DNA ion pair formation are contained within a 28-base pair DNA fragment that has the 22-base pair consensus DNA site at its center. The quantitative data in this report provide base-line thermodynamic data required for detailed investigations of amino acid-base pair and amino acid-phosphate contacts in this protein-DNA complex.     

Effect of salt shock on stability of lambdaimm434 lysogens

The affinities of the bacteriophage 434 repressor for its various binding sites depend on the type and/or concentration of monovalent cations. The ability of bacteriophage 434 repressor to govern the lysis-lysogeny decision depends on the DNA binding activities of the phage's cI repressor protein. We wished to determine whether changes in the intracellular ionic environment influence the lysis-lysogeny decision of the bacteriophage lambda(imm434). Our findings show that the ionic composition within bacterial cells varies with the cation concentration in the growth media. When lambda(imm434) lysogens were grown to mid-log or stationary phase and subsequently incubated in media with increasing monovalent salt concentrations, we observed a salt concentration-dependent increase in the frequency of bacteriophage spontaneous induction. We also found that the frequency of spontaneous induction varied with the type of monovalent cation in the medium. The salt-dependent increase in phage production was unaffected by a recA mutation. These findings indicate that the salt-dependent increase in phage production is not caused by activation of the SOS pathway. Instead, our evidence suggests that salt stress induces this lysogenic bacteriophage by interfering with 434 repressor-DNA interactions. We speculate that the salt-dependent increase in spontaneous induction is due to a direct effect on the repressor's affinity for DNA. Regardless of the precise mechanism, our findings demonstrate that salt stress can regulate the phage lysis-lysogeny switch.     

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

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

Selection and design of high affinity DNA ligands for mutant single-chain derivatives of the bacteriophage 434 repressor

Single-chain repressor RRTRES is a derivative of bacteriophage 434 repressor, which contains covalently dimerized DNA-binding domains (amino acids 1-69) of the phage 434 repressor. In this single-chain molecule, the wild type domain R is connected to the mutant domain RTRES by a recombinant linker in a head-to-tail arrangement. The DNA-contacting amino acids of RTRES at the -1, 1,2, and 5 positions of the α3 helix are T, R, E, S respectively. By using a randomized DNA pool containing the central sequence -CATACAAGAAAGNNNNNTTT-. a cyclic, in vitro DNA-binding site selection was performed. The selected population was cloned and the individual members were characterized by determining their binding affinities to RRTRES. The results showed that the optimal operators contained the TTAC or TTCC sequences in the underlined positions as above, and that the Kd values were in the 1×10-12mol/L1×10-11mol/L concentration range. Since the affinity of the natural 434 repressor to its natural operator sites is in the