Role of the Cro repressor carboxy-terminal domain and flexible dimer linkage in operator and nonspecific DNA binding |
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Authors: | A J Hubbard L P Bracco S J Eisenbeis R B Gayle G Beaton M H Caruthers |
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Institution: | Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215. |
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Abstract: | 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. |
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