Conformational changes necessary for gene regulation by Tet repressor assayed by reversible disulfide bond formation.
We constructed and characterized four Tet repressor (TetR) variants with engineered cysteine residues which can form disulfide bonds and are located in regions where conformational changes during induction by tetracycline (tc) might occur. All TetR mutants show nearly wild-type activities in vivo, and the reduced proteins also show wild-type activities in vitro. Complete and reversible disulfide bond formation was achieved in vitro for all four mutants. The disulfide bond in NC18RC94 immobilizes the DNA reading head with respect to the protein core and prevents operator binding. Formation of this disulfide bond is possible only in the tc-bound, but not in the operator-bound conformation. Thus, these residues must have different conformations when bound to these ligands. The disulfide bonds in DC106PC159' and EC107NC165' immobilize 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 show that the variable loop is located closer to the protein core in the operator-bound conformation and that a movement is necessary for induction. The disulfide bond in RC195VC199' connects alpha-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. Thus, this bond prevents formation of the operator-bound conformation. The detection of conformational changes in three different regions is the first biochemical evidence for induction-associated global internal movements in TetR.
Tiebel
B
B
Lehrstuhl für Mikrobiologie, Biochemie und Genetik der Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Germany.
Aung-Hilbrich
L M
LM
Schnappinger
D
D
Hillen
W
W
D003545:Cysteine; D003553:Cystine; D019281:Dimerization; D004220:Disulfides; D005786:Gene Expression Regulation; D009038:Motion; D009154:Mutation; D009875:Operator Regions, Genetic; D011485:Protein Binding; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D004220:Disulfides; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D003553:Cystine; D003545:Cysteine