The role of the minor groove substituents in indirect readout of DNA sequence by 434 repressor

The sequence of non-contacted bases at the center of the 434 repressor binding site affects the strength of the repressor-DNA complex by influencing the structure and flexibility of DNA (Koudelka, G. B., and Carlson, P. (1992) Nature 355, 89-91). We synthesized 434 repressor binding sites that differ in their central sequence base composition to test the importance of minor groove substituents and/or the number of base pair hydrogen bonds between these base pairs on DNA structure and strength of the repressor-DNA complex. We show here that the number of base pair H-bonds between the central bases apparently has no role in determining the relative affinity of a DNA site for repressor. Instead we find that the affinity of DNA for repressor depends on the absence or presence the N2-NH(2) group on the purine bases at the binding site center. The N2-NH(2) group on bases at the center of the 434 binding site appears to destabilize 434 repressor-DNA complexes by decreasing the intimacy of the specific repressor-DNA contacts, while increasing the reliance on protein contacts to the DNA phosphate backbone. Thus, the presence of an N2-NH(2) group on the purines at the center of a binding site globally alters the precise conformation of the protein-DNA interface.     

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.     

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.     

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.     

DNA-induced conformational changes in bacteriophage 434 repressor

Although bacteriophage 434 repressor binds to its specific DNA sites only as a dimer, formation of the dimers in solution occurs at concentrations three orders of magnitude higher than those needed to bind the 434 operator DNA. Our results suggest that both specific and non-specific DNA induce conformational changes in repressor that lead to formation of repressor dimers. The repressor conformational changes induced by DNA occur at concentrations much lower than those needed for binding of repressor, suggesting that the alternative conformations of repressor persist even if the protein is not in direct contact with DNA. Hence, DNA acts in a "catalytic" fashion to induce a steady-state amount of an alternative repressor conformation that has an enhanced affinity for its specific binding site. These findings suggest that the repressor conformer induced by non-specific DNA is the form of the repressor that is optimized for searching for DNA binding sites along non-specific DNA. Upon finding a binding site, the repressor protein undergoes an additional conformational change that allows it to "lock-on" to its specific site.     

Expression, purification, and functional characterization of the carboxyl-terminal domain fragment of bacteriophage 434 repressor.

The repressor protein of bacteriophage 434 binds to DNA as a dimer of identical subunits. Its strong dimerization is mediated by the carboxyl-terminal domain. Cooperative interactions between the C-terminal domains of two repressor dimers bound at adjacent sites can stabilize protein-DNA complexes formed with low-affinity binding sites. We have constructed a plasmid, pCT1, which directs the overproduction of the carboxyl-terminal domain of 434 repressor. The protein encoded by this plasmid is called CT-1. Cells transformed with pCT1 are unable to be lysogenized by wild-type 434 phage, whereas control cells are lysogenized at an efficiency of 1 to 5%. The CT-1-mediated interference with lysogen formation presumably results from formation of heteromeric complexes between the phage-encoded repressor and the plasmid-encoded carboxyl-terminal domain fragment. These heteromers are unable to bind DNA and thereby inhibit the repressor's activity in promoting lysogen formation. Two lines of evidence support this conclusion. First, DNase I footprinting experiments show that at a 2:1 ratio of CT-1 to intact 434 repressor, purified CT-1 protein prevents the formation of complexes between 434 repressor and its OR1 binding site. Second, cross-linking experiments reveal that only a specific heterodimeric complex forms between CT-1 and intact 434 repressor. This latter observation indicates that CT-1 interferes with 434 repressor-operator complex formation by preventing dimerization and not by altering the conformation of the DNA-bound repressor dimer. Our other evidence is also consistent with this suggestion. We have used deletion analysis in an attempt to define the region which mediates the 434 repressor-CT-1 interaction. CT-1 proteins which have more than the last 14 amino acids removed are unable to interfere with 434 repressor action in vivo.     

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     

Recognition of DNA by single-chain derivatives of the phage 434 repressor: high affinity binding depends on both the contacted and non-contacted base pairs.

Single-chain derivatives of the phage 434 repressor, termed single-chain repressors, contain covalently dimerized DNA-binding domains (DBD) which are connected with a peptide linker in a head-to-tail arrangement. The prototype RR69 contains two wild-type DBDs, while RR*69 contains a wild-type and an engineered DBD. In this latter domain, the DNA- contacting amino acids of thealpha3 helix of the 434 repressor are replaced by the corresponding residues of the related P22 repressor. We have used binding site selection, targeted mutagenesis and binding affinity studies to define the optimum DNA recognition sequence for these single-chain proteins. It is shown that RR69 recognizes DNA sequences containing the consensus boxes of the 434 operators in a palindromic arrangement, and that RR*69 optimally binds to non-palindromic sequences containing a 434 operator box and a TTAA box of which the latter is present in most P22 operators. The spacing of these boxes, as in the 434 operators, is 6 bp. The DNA-binding of both single-chain repressors, similar to that of the 434 repressor, is influenced indirectly by the sequence of the non-contacted, spacer region. Thus, high affinity binding is dependent on both direct and indirect recognition. Nonetheless, the single-chain framework can accommodate certain substitutions to obtain altered DNA-binding specificity and RR*69 represents an example for the combination of altered direct and unchanged indirect readout mechanisms.     

Synthesis of nonlinear DNA-binding peptide with binding specificity determinants close to those of 434 Cro-repressor

Design, synthesis and DNA binding activity of a nonlinear 102 residue peptide are reported. The peptide contains four sequence-specific DNA binding domains of 434 Cro protein. These four domains were linked covalently to a symmetrical carboxyterminal crosslinker that contains four arms each ending with an aliphatic aminogroup. From CD studies we have found that in aqueous buffer in the presence of 20% trifluoroethanol the peptide residues assume alpha helical, beta-sheet and random coiled conformations with an alpha helical content of about 16% at room temperature. The alpha helicity is increased up to 40% in the presence of 40% trifluoroethanol. Upon complex formation between the peptide and DNA a change in the peptide conformation takes place which is consistent with an alpha-beta transition in the DNA binding, helix-turn-helix motif of 434 Cro repressor. Evidently residues present in helices alpha(2) and alpha(3) form a beta hairpin which is inserted in the minor DNA groove. The latter inference is supported by our observations that the peptide can displace minor groove binding antibiotic distamycin A from a complex with poly(dA).poly(dT). As revealed from DNase protection studies the peptide exhibits preferences for binding to operator and pseudooperator sites recognized by 434 Cro repressor. It binds strongly to operator sites OR1, OR2 and OR3 and exhibits a greater affinity for pseudooperator site Op1. From analysis of nucleotide sequences in the strong affinity binding sites for the peptide on DNA a conclusion is drawn that it binds to pseudosymmetrical nucleotide sequences 5'-ACAA(W)nCTGT-3', where W is an arbitrary nucleotide. n is equal to six or seven. In the strongest affinity binding site for the peptide on DNA (Op1) motif 5'-ACAA-3' is replaced by sequence 5'-ACCA-3'. A difference in binding specificity shown by the peptide and 434 Cro protein could be attributed to a flexibility of the connecting chains between DNA-binding domains in the peptide molecule as well as to a replacement of Thr - Ala in the alpha 2 helix. Removal of two residues from the N-terminal end of helix alpha 2 in each of the four DNA binding domains of 434 Cro present in the peptide leads to a loss of binding specificity, although the modified peptide binds to DNA unspecifically.     

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.     

DNA conformational change in Gal repressor-operator complex: involvement of central G-C base pair(s) of dyad symmetry.

Gal repressor dimer binds to two gal operator sites, OE and OI, which are 16 bp long similar sequences with hyphenated dyad symmetries (11,12). Repressor occupation hinders the reactivity of the N7 atoms in the major groups of guanines, located at positions 1, 3 and 8, and the rotational 1', 3' and 8' of the symmetries. We have shown that Gal repressor binding to OE or OI DNA fragments increases the circular dichroism (CD) spectral peak in the 270 to 300 nm range. The CD change is similar to that observed for Lac repressor binding to its operator site (14). It is consistent with a DNA conformational change during complex formation between Gal repressor and OE and OI DNA. The CD spectral change was not observed when the central 8,8' G-C base pairs in the DNA-protein complex were replaced by A-T base pairs, whereas substitution of the 1,1' G-C base pairs do show the accompanying increase in the spectra during repressor binding. The absence of CD change of the Gal repressor complex with DNA mutated at the 8,8' base pairs suggest that the central G-C base pairs are required for the repressor induced conformational change.     

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.     

Dimerization specificity of P22 and 434 repressors is determined by multiple polypeptide segments.

The repressor protein of bacteriophage P22 binds to DNA as a homodimer. This dimerization is absolutely required for DNA binding. Dimerization is mediated by interactions between amino acids in the carboxyl (C)-terminal domain. We have constructed a plasmid, p22CT-1, which directs the overproduction of just the C-terminal domain of the P22 repressor (P22CT-1). Addition of P22CT-1 to DNA-bound P22 repressor causes the dissociation of the complex. Cross-linking experiments show that P22CT-1 forms specific heterodimers with the intact P22 repressor protein, indicating that inhibition of P22 repressor DNA binding by P22CT-1 is mediated by the formation of DNA binding-inactive P22 repressor:P22CT-1 heterodimers. We have taken advantage of the highly conserved amino acid sequences within the C-terminal domains of the P22 and 434 repressors and have created chimeric proteins to help identify amino acid regions required for dimerization specificity. Our results indicate that the dimerization specificity region of these proteins is concentrated in three segments of amino acid sequence that are spread across the C-terminal domain of each of the two phage repressors. We also show that the set of amino acids that forms the cooperativity interface of the P22 repressor may be distinct from those that form its dimer interface. Furthermore, cooperativity studies of the wild-type and chimeric proteins suggest that the location of cooperativity interface in the 434 repressor may also be distinct from that of its dimerization interface. Interestingly, changes in the dimer interface decreases the ability of the 434 repressor to discriminate between its wild-type binding sites, O(R)1, O(R)2, and O(R)3. Since 434 repressor discrimination between these sites depends in large part on the ability of this protein to recognize sequence-specific differences in DNA structure and flexibility, this result indicates that the C-terminal domain is intimately involved in the recognition of sequence-dependent differences in DNA structure and flexibility.     

Design, biochemical, biophysical and biological properties of cooperative antisense oligonucleotides.

Short oligonucleotides that can bind to adjacent sites on target mRNA sequences are designed and evaluated for their binding affinity and biological activity. Sequence-specific binding of short tandem oligonucleotides is compared with a full-length single oligonucleotide (21mer) that binds to the same target sequence. Two short oligonucleotides that bind without a base separation between their binding sites on the target bind cooperatively, while oligonucleotides that have a one or two base separation between the binding oligonucleotides do not. The binding affinity of the tandem oligonucleotides is improved by extending the ends of the two oligonucleotides with complementary sequences. These extended sequences form a duplex stem when both oligonucleotides bind to the target, resulting in a stable ternary complex. RNase H studies reveal that the cooperative oligonucleotides bind to the target RNA with sequence specificity. A short oligonucleotide (9mer) with one or two mismatches does not bind at the intended site, while longer oligonucleotides (21mers) with one or two mismatches still bind to the same site, as does a perfectly matched 21mer, and evoke RNase H activity. HIV-1 inhibition studies reveal an increase in activity of the cooperative oligonucleotide combinations as the length of the dimerization domain increases.