Molecular basis of DNA sequence recognition by the catabolite gene activator protein: detailed inferences from three mutations that alter DNA sequence specificity.

Abstract

Previously, we reported that substitution of Glu-181 of the catabolite gene activator protein (CAP) by lysine, leucine, or valine results in a protein that has specificity for APT base pairs at positions 7 and 16 of the DNA recognition site, rather than GC base pairs as is the case with the wild-type CAP. In this paper, we deduce from these genetic data both (i) the specific chemical interactions by which amino acid side chains at position 181 interact with base pairs 7 and 16 and (ii) the precise alignment between the structures of the CAP and DNA in the intermolecular CAP-DNA complex. Our analysis supports the idea that the two symmetry-related The three-dimensional structures of three proteins that bind to specific DNA sequences have been determined recentlyi.e., the catabolite gene activator protein (CAP) complex with cAMP (1), cro (2), and the amino-terminal fragment of the X repressor (ref. 3; reviewed in ref. 4). Because the structures determined were those of the uncomplexed proteins, efforts to date to elucidate the structures of the protein-DNA complexes have relied on model building (1)(2)(3)(4)(5)(6)(7)(8). The models proposed in the cases of cro and the X repressor and one of four models in the case of CAP exhibit common features. Each model postulates that a pair of 2-fold related ahelices, one from each subunit of the protein, interacts with successive major grooves of right-handed B-type DNA (2, 3, 7). As discussed below, in each instance the a-helix proposed to contact DNA is the second a-helix of a characteristic helix-turn-helix structural motif (9-13). In this paper, we discuss the implications for this analysis of our genetic results (14), which we believe identify a direct contact between an amino acid of a DNA binding protein and the base pair it contacts in the target DNA sequence.

DNA binding specificity and sequence of Xanthomonas campestris catabolite gene activator protein-like protein

“…Amino acid 1 of the recognition helix of CAP (i.e., Arg-180) forms H bonds with the guanine N7 and guanine 06 atoms of G. C at position 5 of the DNA half site (22,29) and determines specificity for G. C at position 5 of the DNA half site (29). Amino acid 2 of the recognition helix of CAP (i.e., Glu-181) forms an H bond with the cytosine N4 atom of G. C at position 7 of the DNA half site (10,12,14,22) and determines specificity for T. A at position 6 of the DNA half site and specificity for G. C at position 7 of the DNA half site (9,10,12,14).…”

“…1 (1,4,19,24) and CLP. In the structure of the CAP-DNA complex, three amino acids of CAP contact DNA base pairs of the DNA half site: i.e., amino acids 1, 2, and 6 of the recognition helix (9,10,12,14,22,29). These amino acids of CAP and the corresponding amino acids of CLP are indicated in boldface type.…”

crp genes of Shigella flexneri, Salmonella typhimurium, and Escherichia coli

“…The sequence of the Escherichia coli K-12 crp gene coding for CAP (2,9), and the structure of the CAP-cAMP complex have been determined, the latter from a 0.29-nm-resolution electron density map (29). By using a genetic approach, we have proposed a model for the CAP-DNA-specific interaction (15,16). Another genetic study has proposed a model describing the effect of cAMP on the activation process by CAP (19).…”

Efficient Tn10 transposition into a DNA insertion hot spot in vivo requires the 5-methyl groups of symmetrically disposed thymines within the hot-spot consensus sequence.

“…The methyl groups in the TnJO insertion-site recognition sequence might make direct contacts with the transposition protein(s), as has been assumed for methyl groups in other cases (23)(24)(25). Interactions of DNA methyl groups with proteins are generally assumed to involve hydrophobic and/or van der Waals interactions, although rigorous proof of such interactions is difficult (see above).…”

“…A positive role for DNA methyl groups has also been proposed for three interactions involving mutant DNAbinding proteins (23)(24)(25). In the latter case, effective binding between a mutant 434 operator and a specific mutant 434 repressor has been proposed to result from a newly generated van der-Waals contact between the methyl group of the (mutant) thymine residue and the methyl group on the alanine side chain of the mutant amino acid (25).…”