Single-chain repressors containing engineered DNA-binding domains of the phage 434 repressor recognize symmetric or asymmetric DNA operators.
Single-chain (sc) DNA-binding proteins containing covalently dimerized N-terminal domains of the bacteriophage 434 repressor cI have been constructed. The DNA-binding domains (amino acid residues 1 to 69) were connected in a head-to-tail arrangement with a part of the natural linker 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 symmetric O(R)1 operator sequence by this sc homodimer was indistinguishable from that of the naturally dimerized repressor in terms of binding 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-contacting amino acid residues of the recognition helix in one of the domains with the corresponding residues of the Salmonella phage P22 repressor c2 resulted in a sc heterodimer of altered specificity. This new heterodimeric molecule recognized an asymmetric, artificial 434-P22 chimeric operator with high affinity. Similar substitutions in both 434 domains have led to a new sc homodimer which showed high affinity binding to a natural, symmetric P22 operator. These findings, supported by both in vitro and in vivo experiments, show that the sc architecture allows for the introduction of independent changes in the binding domains and suggest that this new protein framework could be used to generate new specificities in protein-DNA interaction.
Simoncsits
A
A
International Centre for Genetic Engineering and Biotechnology (ICGEB), Area Science Park, Trieste, Italy.
Chen
J
J
Percipalle
P
P
Wang
S
S
Törö
I
I
Pongor
S
S
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000378:metabolism*; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins