Tet repressor mutants with altered effector binding and allostery

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Abstract
To learn about the correlation between allostery and ligand binding of the Tet repressor (TetR) we analyzed the effect of mutations in the DNA reading head–core interface on the effector specific TetRi2 variant. The same mutations in these subdomains can lead to completely different activities, e.g. the V99G exchange in the wild‐type leads to corepression by 4‐ddma‐atc without altering DNA binding. However, in TetRi2 it leads to 4‐ddma‐atc dependent repression in combination with reduced DNA binding in the absence of effector. The thermodynamic analysis of effector binding revealed decreased affinities and positive cooperativity. Thus, mutations in this interface can influence DNA binding as well as effector binding, albeit both ligand binding sites are not in direct contact to these altered residues. This finding represents a novel communication mode of TetR. Thus, allostery may not only operate by the structural change proposed on the basis of the crystal structures.