CONFORMATIONAL-CHANGES NECESSARY FOR GENE-REGULATION BY TET REPRESSORASSAYED BY REVERSIBLE DISULFIDE BOND FORMATION

We constructed and characterized four Tet repressor (TetR) variants wi th engineered cysteine residues which can form disulfide bonds and are located in regions where conformational changes during induction by t etracycline (tc) might occur. All TetR mutants show nearly wild-type a ctivities in vivo, and the reduced proteins also show wild-type activi ties in vitro. Complete and reversible disulfide bond formation was ac hieved in vitro for all four mutants. The disulfide bond in NC18RC94 i mmobilizes the DNA reading head with respect to the protein core and p revents operator binding. Formation of this disulfide bond is possible only in the tc-bound, but not in the operator-bound conformation. Thu s, these residues must have different conformations when bound to thes e ligands. The disulfide bonds in DC106PC159' and EC107NC165' immobili ze the variable loop between alpha-helices 8 and 9 located near the tc -binding pocket. A faster rate of disulfide formation in the operator- bound conformation and a lack of induction after disulfide formation s how that the variable loop is located closer to the protein core in th e operator-bound conformation and that a movement is necessary for ind uction. The disulfide bond in RC195VC199' connects a-helices 10 and 10 ' of the two subunits in the dimer and is only formed in the tc-bound conformation. The oxidized protein shows reduced operator binding. Thu s, this bond prevents formation of the operator-bound conformation. Th e detection of conformational changes in three different regions is th e first biochemical evidence for induction-associated global internal movements in TetR.