A 30-amino-acid segment of C/EBP, a newly discovered enhancer binding protein, shares notable sequence similarity with a segment of the cellular Myc transforming protein. Display of these respective amino acid sequences on an idealized alpha helix revealed a periodic repetition of leucine residues at every seventh position over a distance covering eight helical turns. The periodic array of at least four leucines was also noted in the sequences of the Fos and Jun transforming proteins, as well as that of the yeast gene regulatory protein, GCN4. The polypeptide segments containing these periodic arrays of leucine residues are proposed to exist in an alpha-helical conformation, and the leucine side chains extending from one alpha helix interdigitate with those displayed from a similar alpha helix of a second polypeptide, facilitating dimerization. This hypothetical structure is referred to as the "leucine zipper," and it may represent a characteristic property of a new category of DNA binding proteins.

https://science.sciencemag.org/content/sci/240/4860/1759.full.pdf

The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins

Thermodynamic studies have demonstrated the central importance of a large negative heat capacity change (delta C degree assoc) in site-specific protein-DNA recognition. Dissection of the large negative delta C degree assoc and the entropy change of protein-ligand and protein-DNA complexation provide a thermodynamic signature identifying processes in which local folding is coupled to binding. Estimates of the number of residues that fold on binding obtained from this analysis agree with structural data. Structural comparisons indicate that these local folding transitions create key parts of the protein-DNA interface. The energetic implications of this "induced fit" model for DNA site recognition are considered.

https://science.sciencemag.org/content/sci/263/5148/777.full.pdf

Coupling of local folding to site-specific binding of proteins to DNA

The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer.

Molecular biologists are increasingly faced with the problem of assigning a function to genes that have been cloned. A new approach to this problem involves the manipulation of the cloned gene to create what are known as 'dominant negative' mutations. These encode mutant polypeptides that when overexpressed disrupt the activity of the wild-type gene. There are many precedents for this kind of behaviour in the literature--some oncogenes might be examples of naturally occurring dominant negative mutations.

Functional inactivation of genes by dominant negative mutations

The 3 angstrom resolution crystal structure of the Escherichia coli catabolite gene activator protein (CAP) complexed with a 30-base pair DNA sequence shows that the DNA is bent by 90 degrees. This bend results almost entirely from two 40 degrees kinks that occur between TG/CA base pairs at positions 5 and 6 on each side of the dyad axis of the complex. DNA sequence discrimination by CAP derives both from sequence-dependent distortion of the DNA helix and from direct hydrogen-bonding interactions between three protein side chains and the exposed edges of three base pairs in the major groove of the DNA. The structure of this transcription factor--DNA complex provides insights into possible mechanisms of transcription activation.

/pdf/crystal-structure-of-a-cap-dna-complex-the-dna-is-bent-by-90-1s4euserja.pdf

Crystal structure of a CAP-DNA complex: the DNA is bent by 90 degrees.

The three-dimensional structure of the 66-amino acid cro repressor protein of bacteriophage λ suggests how it binds to its operator DNA. We propose that a dimer of cro protein is bound to the B-form of DNA with the 2-fold axis of the dimer coincident with the 2-fold axis of DNA. A pair of 2-fold-related α-helices of the represser, lying within successive major grooves of the DNA, seem to be a major determinant in recognition and binding. In addition, the C-terminal residues of the protein, some of which are disordered in the absence of DNA, appear to contribute to the binding.

Structure of the cro repressor from bacteriophage λ and its interaction with DNA

The structures of three proteins that regulate gene expression have been determined recently and suggest how these proteins may bind to their specific recognition sites on the DNA. One protein (Cro) is a repressor of gene expression, the second (CAP) usually stimulates gene expression, and the third (lambda repressor) can act as either a repressor or an activator. The three proteins contain a substructure consisting of two consecutive alpha helices that is virtually identical in each case. Structural and amino acid sequence comparisons suggest that this bihelical fold occurs in a number of proteins that regulate gene expression, and is an intrinsic part of the DNA-protein recognition event. The modes of repression and activation by Cro and lambda repressor are understood reasonably well, but the mode of action of CAP is still unclear.

https://science.sciencemag.org/content/sci/221/4615/1020.full.pdf

DNA-binding proteins

We measured quantitatively the binding affinities of purified Cro repressor to the chemically synthesized wild-type and mutant OR1 operators, consisting of all three possible base-pair substitutions and of thymine to uracil substitutions at each base-pair position of the 17-base-pair operator sequence. The sequence-specific interactions between Cro repressor and the operator DNA occur at the symmetrically disposed outer 7-base-pair positions of each half operator and at the central base-pair position. The binding of Cro is almost symmetrical with respect to the pseudo-twofold symmetry of the binding site. The binding free energy changes calculated from the affinity changes are mostly additive for specific Cro binding. Also the binding affinities of Cro to the operators or any other DNA sequences can be predicted by simple addition of free energy changes of single base substitutions. We isolated cro mutants by site-directed mutagenesis and studied their DNA binding to the wild-type and base-substituted mutant operators. The sequence-specific contacts derived from such studies are significantly different from the models proposed by Ohlendorf et al. [Ohlendorf, D. H., Anderson, W. F., Takeda, Y. & Matthews, B. W. (1982) Nature (London) 298, 719-723] and by Hochschild et al. [Hochschild, A., Douhan, J., III, & Ptashne, M. (1986) Cell 47, 807-816].

https://www.pnas.org/content/pnas/86/2/439.full.pdf

Analysis of the sequence-specific interactions between Cro repressor and operator DNA by systematic base substitution experiments.

Results of systematic base-substitution experiments suggest that the lambda repressor dimer, made of identical subunits, recognizes the "pseudo(2-fold)symmetric" operator sequence asymmetrically. Base substitutions within the consensus half of the operator affect binding more than base substitutions within the nonconsensus half of the operator. Furthermore, changing the nonconsensus base pairs to the consensus base pairs does not increase, but decreases, binding. Evidently, the two subunits of the lambda repressor dimer bind to the two halves of the operator differently. This is consistent with the recently determined crystal structure of the complex, which shows that the relative positioning of the amino acids to the DNA bases are slightly different in the two halves of the operator. The sequence-specific interactions indicated by the systematic base-substitution experiments correlate well with the locations of the specific contacts found in the complex. Thus, the amino acids of lambda repressor, mainly of alpha 3-helix and the N-terminus arm, seem to directly read-out the DNA sequence by forming specific hydrogen bonds and hydrophobic contacts to the DNA bases. The observed asymmetric recognition suggests that no recognition code governs amino acids and DNA bases in protein-DNA interactions.

https://www.pnas.org/content/pnas/86/17/6513.full.pdf

Lambda repressor recognizes the approximately 2-fold symmetric half-operator sequences asymmetrically.

The structure of a complex of bacteriophage lambda Cro protein with a 17-base-pair operator has been determined at 3.9-A resolution. Isomorphous derivatives obtained by the synthesis of site-specific iodinated DNA oligomers were of critical importance in solving the structure. The crystal structure contains three independent Cro-operator complexes that have very similar, although not necessarily identical, conformations. In the complex, the protein dimer undergoes a large conformational change relative to the crystal structure of the free protein. One monomer rotates by about 40 degrees relative to the other, this being accomplished primarily by a twisting of the two beta-sheet strands that connect one monomer with the other. In the complex, the DNA is bent by about 40 degrees into the shape of a boomerang but maintains essentially Watson-Crick B-form. In contrast to other known protein-DNA complexes, the DNA is not stacked end-to-end. The structure confirms the general features of the model previously proposed for the interaction of Cro with DNA.

http://www.pnas.org/content/87/20/8165.full.pdf

Yoshinori Takeda

Papers

Structure of the cro repressor from bacteriophage λ and its interaction with DNA

DNA-binding proteins

Analysis of the sequence-specific interactions between Cro repressor and operator DNA by systematic base substitution experiments.

Lambda repressor recognizes the approximately 2-fold symmetric half-operator sequences asymmetrically.

Protein-DNA conformational changes in the crystal structure of a lambda Cro-operator complex