J. Biol. Chem., Vol. 261, Issue 19, 8608-8616, 07, 1986
Different interactions used by Cro repressor in specific and nonspecific DNA binding
Y Takeda, JG Kim, CG Caday, E Steers Jr, DH Ohlendorf, WF Anderson and BW Matthews
The mode of interaction of Cro repressor with specific and nonspecific
sites on DNA was explored by chemical modification and protection of lysine
and tyrosine residues. Cro has 8 lysines. In the presence of DNA, lysines
32 and 56 are fully protected and lysines 21, 62, and 63 are partially
protected from alkylation. However, the terminal amino group and lysines 8,
18, and 39 are not protected. Location of the protected and unprotected
lysines on the three-dimensional Cro structure defines a DNA-binding
region. The results provide direct experimental support for a mode of
interaction between Cro and DNA, in which Cro buries its 2-fold related
alpha-helices in consecutive DNA major grooves (Anderson, W. F., Ohlendorf,
D. H., Takeda, Y., and Matthews, B. W. (1981) Nature 290, 754-758;
Ohlendorf, D. H., Anderson, W. F., Fisher, R. G., Takeda, Y., and Matthews,
B. W. (1982) Nature 298, 718-723). In the model, the carboxyl-terminal part
of Cro was tentatively presumed to interact with the DNA minor groove.
Protection of lysines 62 and 63 confirms the involvement of the carboxyl
terminus in DNA binding. Although nonspecific and specific DNA protect the
same lysine residues, there are differences in the nature of the
interaction of Cro with nonspecific and specific DNA. Cro-nonspecific DNA
interaction is salt-sensitive, suggesting that the interaction is
predominantly electrostatic. On the other hand, Cro-specific DNA
interaction is salt-resistant, suggesting that the interaction may include
nonelectrostatic components (hydrogen bonds and hydrophobic interactions)
as well. Protection experiments of tyrosine residues (against iodination)
suggest that the conformation of Cro repressor changes in two stages:
first, when Cro binds at nonspecific sites, and, second, when Cro binds to
specific sites on DNA.