Role of the Cro repressor carboxy-terminal domain and flexible dimer linkage in operator and nonspecific DNA binding.
A series of mutations comprising single and multiple substitutions, deletions, and extensions within the carboxy-terminal domain of the bacteriophage lambda Cro repressor have been constructed. These mutations generally affect the affinity of repressor for specific and nonspecific DNA. Additionally, substitution of the carboxy-terminal alanine with several amino acids capable of hydrogen-bonding interactions leads to improved specific binding affinities. A mutation is also described whereby cysteine links the two Cro monomers by a disulfide bond. As a consequence, a significant improvement in nonspecific binding and a concomitant reduction in specific binding are observed with this mutant. These results provide evidence that the carboxy terminus of Cro repressor is an important DNA binding domain and that a flexible connection between the two repressor monomers is a critical factor in modulating the affinity of wild-type repressor for DNA.
Hubbard
A J
AJ
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
Bracco
L P
LP
Eisenbeis
S J
SJ
Gayle
R B
RB
Beaton
G
G
Caruthers
M H
MH
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009875:Operator Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics*
D012097:Repressor Proteins; D004247:DNA