Interaction of the Bacillus subtilis phage phi 105 repressor DNA: a genetic analysis.

The Bacillus subtilis phage phi 105 repressor specifically recognizes a 14-bp operator sequence which does not exhibit 2-fold rotational symmetry. To facilitate a genetic analysis of this sequence-dependent DNA binding a B. subtilis strain was constructed in which mutations affecting the phi 105 repressor-operator interaction cause a selectable phenotype, chloramphenicol resistance. After in vivo mutagenesis, we isolated and mapped 22 different mutations in the repressor coding sequence, 15 of which are missense substitutions. These are exclusively located in the N-terminal part (positions 1-43) of the 144 residue long polypeptide. Two nonsense mutants, at positions 70 and 89, respectively, still show partial repressor activity. These data suggest that the phi 105 repressor consists of at least two independently folding structural domains, of which the N-terminal is involved in operator binding. Twelve missense mutations are clustered in a region extending from Gln-18 to Arg-37, which we propose to be the DNA-binding alpha-helix--beta-turn--alpha-helix motif, common to all lambda Cro-like repressors. The second ('recognition') helix shows significant homology with the corresponding sequence in Tn3 resolvase, and there is also a striking similarity between the phi 105 operator and the consensus sequence for a Tn3 res half-site. Based on these observations, and on the previously isolated phi 105 0c mutants, we tentatively assign some specific contacts between base pairs from the first half of a phi 105 operator site and amino acids from the repressor's 'recognition helix'.     

Specificity of the interaction between lambda cro repressor protein and operator DNA fragments

Cro repressor protein is known to interact with specific sites in the operator DNA. The cro protein of lambda phage was isolated and the mode of its interaction with three different DNA fragment, lambda-OR3 17mer, phi 80-OR2 19mer and CAP binding site 22-mer, were examined by the use of proton NMR. Some of the imino proton resonances of lambda-OR3 shifted and were broadened remarkably on addition of lambda-cro protein, which indicated the induction of conformational change with complexation. In the spectrum of phi 80-OR2 which has a six base pair sequence common to lambda-OR3 the signals of the common base pairs revealed slight shifts on addition of lambda-cro protein. The imino proton signals of the CAP site DNA, however, did not show any change at all on mixing with lambda-cro. Combining the data of photo CIDNP of lambda-cro, we could postulate the mode of interaction between lambda-cro repressor and operator DNA.     

Purification and characterization of repressor of temperate S. aureus phage phi11

To gain insight into the structure and function of repressor proteins of bacteriophages of gram-positive bacteria, repressor of temperate Staphylococcus aureus phage phi11 was undertaken as a model system here and purified as an N-terminal histidine-tagged variant (His-CI) by affinity chromatography. A approximately 19 kDa protein copurified with intact His-CI (approximately 30 kDa) at low level was resulted most possibly due to partial cleavage at its Ala-Gly site. At approximately 10 nM and higher concentrations, His-CI forms significant amount of dimers in solution. There are two repressor binding sites in phi11 cI-cro intergenic region and binding to two sites occurs possibly by a cooperative manner. Two sites dissected by HincII digestion were designated operators O(L) and O(R), respectively. Equilibrium binding studies indicate that His-CI binds to O(R) with a little more strongly than O(L) and binding species is probably dimeric in nature. Interestingly His-CI binding affinity reduces drastically at elevated temperatures (32-42 degrees C). Both O(L) and O(R) harbor a nearly identical inverted repeat and studies show that phi11 repressor binds to each repeat efficiently. Additional analyses indicate that phi11 repressor, like lambda repressor, harbors an N-terminal domain and a C-terminal domain which are separated by a hinge region. Secondary structure of phi11 CI even nearly resembles to that of lambda, phage repressor though they differ at sequence level. The putative N-terminal HTH (helix-turn-helix) motif of phi11 repressor belongs to the HTH -XRE-family of proteins and shows significant identity to the HTH motifs of some proteins of evolutionary distant organisms but not to HTH motifs of most S. aureus phage repressors.     

Study of the structure of phage lambda operator OR1 using two-dimensional 1H-NMR-spectroscopy

NOESY spectroscopy at 500 HMz was employed to assign resonances of nonexchangeable protons in the 1H NMR spectrum of the synthesized 17 base pair duplex of deoxyribonucleotides comprising the OR1 operator, one of the specific binding sites for the bacteriophage lambda cro repressor. A pure absorption spectrum that was obtained by the phase sensitive detection technique allowed to perform a resonance assignment of base and deoxyribose protons, excepting H-5'- and H-5"-protons, on the basis of a single NOESY experiment, mixing time 200 ms. The sequential assignment strategy for 2D NMR spectra of oligonucleotides was used for this purpose. The data obtained and the previous results on OR3 are discussed in the view of the conformation of operators in solution. It is shown that both duplexes exist in the DNA B-form with its intrinsic anti conformation of the nucleotides. Conformation of DNA segments with identical primary structures are similar, and local deviations from the classical B-form are determined by a nucleotide sequence.     

Proton-linked contributions to site-specific interactions of lambda cI repressor and OR

The effects of proton activity on the site-specific interactions of cI repressors with operator sites OR were studied by using DNase I footprint titration. Individual-site binding isotherms were obtained for the binding of repressor to each site of wild-type OR and of mutant operators in which binding to some sites is eliminated. The Gibbs energies for binding and for cooperativity (in every operator configuration) were determined at each pH (range 5-8). The proton-linked effects clearly account for a significant fraction of the difference in affinities for the three operator sites. The most dramatic effects on the repressor-operator binding interactions are at acid pH, and therefore do not involve the basic groups in the repressor N-terminal arm known to contact the DNA. Also, the proton-linked effects are different at the three operator sites as indicated by significantly different derivative relationships, partial derivative of ln k versus partial derivative of ln aH = net proton absorption (delta nu bar(H)). These results implicate ionizable repressor groups which may not contact the DNA and conformational differences between the three repressor-operator site complexes as being important components to the mechanism of site specificity. The extensive data base generated by these studies was also used to reevaluate the traditional models used to describe cooperativity in this system. The results confirm the lack of significant cooperative interaction between OR1 and OR3 at all conditions. However, the data for some experimental conditions are clearly inconsistent with the (selection) rule, that cooperative interaction between OR2 and OR3 is eliminated by ligation at OR1.     

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.     

Interactions between lac repressor protein and site-specific bromodeoxyuridine-substituted operator DNA. Ultraviolet footprinting and protein-DNA cross-link formation

Specific contacts between the lac repressor and operator have been explored using 5-bromodeoxyuridine-substituted DNA. Substitution of BrdU for single thymidine positions in a synthetic 40-base pair operator provides substrate for ultraviolet irradiation; upon irradiation, strand scission occurs at the BrdU residues. When bound, lac repressor protein provides protection against UV-induced breakage depending on the nature of the sites and type of interaction. We have confirmed 13 unique sites of inducer-sensitive protection along the operator sequence using this method compared to complete substitution with BrdU; differences were observed at two positions for singly substituted versus completely substituted DNAs (Ogata, R., and Gilbert, W. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 4973-4976). The ability of these photosensitive DNAs to form short range cross-links to bound protein has been used to determine the efficiency with which cross-linked protein-DNA complexes are generated at each individual site of BrdU substitution. Five sites of high efficiency cross-linking to the repressor protein have been identified. At one site, cross-linking without protection from strand scission was observed; this result suggests an unusual mechanism of strand scission and/or cross-linking at this site. Comparison of the UV protection results and the cross-linking data show that these processes provide complementary tools for identifying and analyzing individual protein-DNA contacts.     

Cooperative protein-DNA interactions: effects of KCl on lambda cI binding to OR.

The effects of monovalent salt activity on the site-specific and cooperative interactions of cI repressor with its three operator sites OR were studied by using quantitative DNase I footprint titration methods. Individual-site binding isotherms were obtained for binding repressor dimers to each site of wild-type OR and to mutant operator templates in which binding to one or two sites has been eliminated. The standard Gibbs energies for intrinsic binding, delta G1, delta G2, and delta G3, and cooperative interactions, delta G12 and delta G23, were determined at each condition (range 50-200 mM KCl). It is found that the dimer affinity for each of the three sites increases as [KCl] decreases, a striking result given that the monomer-dimer equilibrium shifts toward monomer formation under identical solution conditions [Koblan, K. S., & Ackers, G. K. (1991) Biochemistry (preceding paper in this issue)]. The magnitudes of ion-linked effects are found to differ at the three operator sites, while the intrinsic interaction binding free energies for sites OR1 and OR3 change in parallel over the entire range of [KCl]. The KCl dependencies at OR1 and OR3 represent the average release of 3.7 +/- 0.6 and 3.8 +/- 0.6 apparent ions, respectively. By contrast, the KCl dependency of OR2 binding corresponds to the displacement of 5.2 +/- 0.7 apparent ions. The ability of cI repressor to discriminate between the three operator sites thus appears linked to ion binding/release reactions.     

Nucleotide sequence of the operators of lambda ultravirulent mutants.

The nucleotide sequence of the operators of ultravirulent mutants of lambda, able to grow on host cells with elevated repressor levels, was determined. It appears that ultravirulence in lambda requires multiple mutational events at the operator sequences. OL1, OL2, and OL3 operator sites are the target of mutational changes in ultravirulent phages indicating that these sites participate in vivo in repression of the PL promoter. No changes were found in the OR3 sequence, in contrast there is a mutation in OR2 and two mutations in OR1, in both lambda 668 and lambda 2668 phages. This mutated operator structure accounts for the constitutive expression of their PR promoter either in cells overproducing the lambda repressor or in cells overproducing the cro gene product. A model of the structure of the lambda operator site is proposed. The nucleotide sequence in each site can be divided into two functionally different subsets, one of which is recognized by the repressor while the other stabilizes the repressor-operator interaction.     

An alkaline phosphatase protection assay to investigate trp repressor/operator interactions

We have used an alkaline phosphatase protection assay to investigate the interaction of the trp repressor with its operator sequence. The assay is based on the principle that the trp repressor will protect a terminally 5'-32P-labeled operator DNA fragment from attack by alkaline phosphatase. The optimal oligonucleotide for investigating the trp repressor/operator interaction extends two base pairs from each end of the genetically defined target sequence predicted by in vivo studies [Bass et al. (1987) Genes Dev. 1, 565-572]. The assay works well over a 10,000-fold range of protein/DNA affinity and is used to show that the corepressor, L-tryptophan, causes the liganded repressor to bind a 20 base pair trp operator duplex 6400 times more strongly than the unliganded aporepressor. The affinity of the trp repressor for operators containing symmetrical mutations was interpreted in terms of the trp repressor/operator crystal structure as follows: (1) Direct hydrogen bonds with the functional groups of G-9 of the trp operator and the side chain of Arg 69 of the trp repressor contribute to DNA-binding specificity. (2) G-6 of the trp operator is critical for DNA-binding specificity probably because of the two water-mediated hydrogen bonds between its functional groups and the N-terminus of the trp repressor's E-helix. (3) Sequence-dependent aspects of the trp operator's conformation help stabilize the trp repressor/operator complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical synthesis and biochemical reactivity of bacteriophage lambda PR promoter

By a combination of chemical and enzymatic methods, a 75 base pair DNA duplex containing the sequence of the lambda PR promoter including the OR1 and OR2 cI repressor binding sites was synthesized. The solid support phosphite triester procedure (Caruthers, M. H. et al., Cold Spring Harbor Symposia on Quantitative Biology XLVII, in press) was used for the synthesis of oligonucleotides comprising the sequence. We report here an adaptation of the method of DNA synthesis in test tubes. Assembly of the oligonucleotides involved the use of T4 polynucleotide kinase and T4 DNA ligase. We show that the synthetic DNA is recognized by RNA polymerase and cI repressor in a manner identical to the same control region contained on a restriction fragment isolated from bacteriophage lambda DNA. Our synthetic approach using chemically synthesized promoter variants is thus suitable for studies probing the function of promoters.