Electron microscopic study of the repressor of bacteriophage λ and its interaction with operator DNA

The structure of purified phage λ repressor has been examined by high resolution electron microscopy. The repressor molecule appears predominantly as a tetramer of about 95 Å × 120 Å. We have proposed a model to account for the variety of aspects seen on the electron micrographs. Spreading DNA without protein film and use of uranyl formate staining allowed the simultaneous visualization of the DNA and the structure of the repressor molecule bound to it. Mapping the positions of λ repressor bound to whole λ DNA shows preferential binding to the region containing the operators. At high resolution multiple binding of repressor to the operator can be demonstrated. Depending on the amount of repressor present, rows of one to four repressor tetramers are seen on the DNA, confirming the model of the operator containing four binding sites for repressor. The bound repressor can consequently protect against nuclease digestion of operator pieces of approximately 30, 57, 87 and 111 base-pairs. The isolated operator appears in the electron microscope as short double-stranded DNA fragments which can be shown to rebind repressor.     

Water release associated with specific binding of gal repressor.

Water release coupled to the association of gal repressor with DNA is measured from the sensitivity of the binding constant to the solution osmotic pressure, using neutral solutes that are typically excluded from polar protein and DNA surfaces. Differences in water release for binding of repressor to different sequences are linked with differences in specificity and binding energies. With sucrose, the specific binding of repressor to operator sequences is accompanied by the release of 130 water molecules. No water release is seen for the weak, non-specific binding of repressor to poly(dI-dC).(dI-dC). A difference in the release of six water molecules is seen even for the binding of gal repressor to two different operator sequences that differ in affinity by only a factor of two.     

A code governing specific binding of regulatory proteins to DNA and structure of stereospecific sites of regulatory proteins]

A model is proposed for the structure of stereospecific sites in regulatory proteins. On its basis a possible code is suggested that governs the binding of regulatory proteins at specific control sites on DNA. Stereospecific sites of regulatory proteins are assumed to contain pairs of antiparallel polypeptide chain segments which form a right-hand twisted antiparallel beta-sheet, with single-stranded regions at the ends of the beta-structure. The model predicts that binding reaction between a regulatory protein and double-helical DNA is a cooperative phenomenon and is accompanied by significant structural alteration at the stereospecific site of the protein. Half of hydrogen bonds normally existing in beta-structure are broken upon complex formation with DNA and a new set of hydrogen bonds is formed between polypeptide amide groups and DNA base pairs. In a stereospecific site, one chain (t-chain) is attached through hydrogen bonds to the carbonyl oxygens of pyramides and N3 adenines lying in one DNA strand, while the second polypeptide chain (g chain) is hydrogen bonded to the 2-amino groups of guanine residues lying in the opposite DNA strand. The amide groups serve as specific reaction sites being hydrogen bond acceptors in g-chain and hydrogen bond donors in t-chain. The single-stranded portions of t- and g-chains lying in neighbouring subunits of regulatory protein interact with each other forming deformed beta-sheets. The recognition of regulatory sequences by proteins is based on the structural complementarity between stereospecific sites of regulatory proteins and base pairs sequences at the control sites. An essential feature of these sequences is the asymmetrical distribution of guanine residues between the two DNA strands. The code predicts that there are six fundamental amino acid residues (serine, threonine, asparagine, histidine, glutamine and cysteine) whose sequence in stereospecific site determines the base pair sequence to which a given regulatory protein would bind preferentially. The code states a correspondence between four amino acid residues at the stereospecific site of regulatory protein with the two residues being in t- and g-segments, respectively, and AT(GC) base pair at the control site. It is thus possible to determine which amino acid residues in the repressor and which base pairs in the operator DNA are involved in specific interactions with each other, as exemplified by lac repressor binding to lac operator.     

Tet repressor-tet operator contacts probed by operator DNA-modification interference studies

Contacts between tet operator DNA and Tet repressor protein are characterized by modification interference studies. The modified DNA fragments are separated into fractions with high, intermediate and low affinities for Tet repressor by polyacrylamide gel electrophoresis. Ethylation of the phosphates with N-ethylnitrosourea reveals 12 contacts of a repressor dimer to tet operator. Eight of these contacts appear to be important for Tet repressor binding, as judged by the strong interference at these positions, while four contacts are probably less important. All of the phosphate contacts are located on the same side of the B-DNA structure. The sequences of tet operators proposed to interact with the recognition alpha-helix of Tet repressor are TCTATC in three cases and CCTATC in one case. After methylation of N-7 with dimethylsulfate, strong interference is observed at the guanine residues at positions +/- 2. None of the N-7 functions of other guanine residues seems to be involved in tight contacts to Tet repressor. Tet repressor subunits form identical phosphate and guanine N-7 contacts with each half side of the two tet operators indicating twofold dyad symmetry of the complexes. Attempts to analyze the methylation interference at the adenine N-3 sites reveal different results for the operators. Modification of DNA fragments with diethylpyrocarbonate yields hypersensitive sites in the tet operators, indicating different local DNA structures. Carbethoxylation interference studies confirm the contacts at the purines found by methylation interference. All of the sequence-specific protein-DNA contacts detected in this study are centered at the inside four base-pairs in each tet operator half side. The contacts are discussed with respect to the structure of the repressor-operator complex.     

DNA recognition by the helix-turn-helix motif: investigation by laser Raman spectroscopy of the phage lambda repressor and its interaction with operator sites OL1 and OR3.

The lambda repressor provides a model system for biophysical studies of DNA recognition by the helix-turn-helix motif. We describe laser Raman studies of the lambda operator sites OL1 and OR3 and their interaction with the DNA-binding domain of lambda repressor (residues 1-102). Raman spectra of the two DNA sites exhibit significant differences attributable to interstrand purine-purine steps that differ in the two oligonucleotides. Remarkably, the conformation of each operator is significantly and specifically altered by repressor binding. Protein recognition, which involves hydrogen-bond formation and hydrophobic contacts in the major groove, induces subtle changes in DNA Raman bands of interacting groups. These include (i) site-specific perturbations to backbone phosphodiester geometry at AT-rich domains, (ii) hydrophobic interaction at thymine 5CH3 groups, (iii) hydrogen bonding to guanine 7N and 6C = O acceptors, and (iv) alterations in sugar pucker within the C2'-endo (B-DNA) family. These perturbations differ between aqueous OL1 and OR3 complexes of repressor, indicating that protein binding in solution determines the precise DNA conformation. The overall structure of the lambda domain is not greatly perturbed by binding to either OL1 or OR3, in accord with X-ray studies of other complexes. However, Raman markers indicate a change in hydrogen bonding of the OH group of tyrosine-22, which is a hydrogen-bond acceptor in the absence of DNA but a combined donor and acceptor in the OL1 complex; yet, Y22 hydrogen bonding is not altered in forming the OR3 complex. The present results demonstrate qualitatively different and distinguishable modes of interaction of the lambda repressor DNA-binding domain with operators OL1 and OR3 in solution. This application of laser Raman spectroscopy to a well-characterized system provides a prototype for future Raman studies of other DNA-binding motifs under physiological conditions.     

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.