SINGLE-CHAIN REPRESSORS CONTAINING ENGINEERED DNA-BINDING DOMAINS OF THE PHAGE-434 REPRESSOR RECOGNIZE SYMMETRICAL OR ASYMMETRIC DNA OPERATORS

Single-chain (sc) DNA-binding proteins containing covalently dimerized N-terminal domains of the bacteriophage 434 repressor cl have been co nstructed. The DNA-binding domains (amino acid residues 1 to 69) were connected in a head-to-tail arrangement with a part of the natural lin ker sequence that connects the N and C-terminal domains of the intact repressor. Compared to the isolated N-terminal DNA-binding domain, the sc molecule showed at least 100-fold higher binding affinity in vitro and a slightly stronger repression in vivo. The recognition of the sy mmetric O(R)1 operator sequence by this sc homodimer was indistinguish able from that of the naturally dimerized repressor in terms of bindin g affinity, DNase I protection pattern and in vivo repressor function. Using the new, sc framework, mutant proteins with altered DNA-binding specificity have also been constructed. Substitution of the DNA-conta cting amino acid residues of the recognition helix in one of the domai ns with the corresponding residues of the Salmonella phage P22 repress or c2 resulted in a sc heterodimer of altered specificity. This new he terodimeric molecule recognized an asymmetric, artificial 434-P22 chim eric operator with high affinity. Similar substitutions in both 434 do mains have led to a new sc homodimer which showed high affinity bindin g to a natural, symmetric P22 operator. These findings, supported by b oth in vitro and in vivo experiments, show that the sc architecture al lows for the introduction of independent changes in the binding domain s and suggest that this new protein framework could be used to generat e new specificities in protein-DNA interaction. (C) 1997 Academic Pres s Limited.