No abstract

Both the amino‐terminal and the carboxy‐terminal domain of the LexA repressor have been purified using the LexA protein autodigestion reaction at alkaline pH, which leads to the same specific products as the physiological RecA‐catalyzed proteolysis of repressor. We show by circular dichroism (c.d) that, upon non‐specific binding to DNA, the purified amino‐terminal domain induces a very similar if not identical conformational change of the DNA as does the entire repressor. The positive c.d. signal increases approximately 3‐fold if the DNA lattice is fully saturated with protein. Further, the amino‐terminal domain of the LexA protein binds specifically to the operator of the recA gene, producing qualitatively the same effects on the methylation pattern of the guanine bases by dimethylsulfate as the entire repressor, consisting of a methylation inhibition effect at four distal operator guanines and a slight enhancement at the central bases. The spacing between these contacts suggests that LexA does not bind to the operator along the same face of the DNA helix. As shown by c.d. studies the amino‐terminal domain harbours a substantial amount of residues in alpha‐helical conformation, a prerequisite for DNA recognition via a helix‐‐turn‐‐helix structural motif as proposed for many other regulatory proteins.

The ability of the isolated carboxy-terminal domain of the LexA repressor of Escherichiu coli to form dimers and tetramers has been investigated by equilibrium ultracentrifugation. This domain, that comprises the amino acids 85-202, is readily purified after self-cleavage of the LexA repressor at alkaline pH. It turns out that the carboxy-terminal domain forms dimers and tetramers essentially as the entire LexA repressor. The corresponding association constants were determined after non-linear least squares fitting of the experimental concentration distribution. A dimer association constant of Kz = 3 x 104 M-r and a tetramer association constant of K, = 2 x 104 M-r have been determined. Similar measurements on the entire LexA repressor [(1985) Biochemistry 24, 2812-28181 gave values of Kz = 2.1 x lo4 M-r and & = 7.7 x 104 M-r. Within experimental error the dimer formation constant of the carboxy-terminal domain may be considered to be the same as that of the entire repressor whereas the isolated domain forms tetramers slightly less efficiently. It should be stressed that the potential error in K4 is higher than that in K2. The overall conclusion is that the two structural domains of LexA have also well-defined functional roles: the amino-terminal domain interacts with DNA and the carboxy-terminal domain is involved in dimerisation reinforcing in this way the binding of the LexA repressor to operator DNA.