THE ROLE OF THE VARIABLE REGION IN TET REPRESSOR FOR INDUCIBILITY BY TETRACYCLINE

A set of deletions and substitutions to alanine was introduced into th e loop separating helices alpha 8 and alpha 9 of Tn10 Tet repressor (T etR). This region appears as an unstructured loop in the crystal struc ture of the TetR(D).([Mg-tc](+))(2) complex and is the only internal s egment of variable length in an alignment of Tet repressors from seven different resistance determinants. In vivo analysis of 10 mutants sho ws that this loop is important for inducibility by tetracycline (tc), whereas DNA binding is not or only marginally affected, All deletions have an induction-deficient TetR(S) phenotype, but the corresponding s ubstitutions do not or only slightly affect inducibility. The purified mutant TetR proteins have a reduced affinity for tc in vitro that cor relates with their lack of inducibility. The association rate of [Mg-t c](+) to the TetR mutants is enhanced. Since none of the mutated resid ues contacts tc directly in the crystal structure, we propose that the length of the loop is important for the structural transition between a closed, tc binding and an open, operator binding conformation of Te tR. We propose that the deletions in the loop shift the equilibrium be tween both forms toward the open, operator binding conformation.