Activity reversal of Tet repressor caused by single amino acid exchanges.
We explore by extensive mutagenesis regions in the sequence allowing reversal of the allosteric response of Tet repressor. The wild type requires anhydrotetracycline for induction. About 100 mutants are presented, which, in contrast, require the drug for repression. Their mutations are clustered at the interface of the DNA- and inducer-binding domains. This interface consists of a central hydrophobic region surrounded by several hydrogen bonds. While most of the mutants described here contain two to five mutations, we found five positions in this region of TetR, at which single amino acid exchanges lead to activity reversal. They may disrupt the hydrogen-bonding network bordering the domain interface. We assume that the mutations cause a repositioning of the DNA reading head with respect to the effector binding core so that the same conformational change can result in opposite activities.
Scholz
Oliver
O
Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie, Biochemie und Genetik, Friedrich-Alexander Universität Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Germany.
Henssler
Eva-Maria
EM
Bail
Johannes
J
Schubert
Peter
P
Bogdanska-Urbaniak
Joanna
J
Sopp
Sabine
S
Reich
Marco
M
Wisshak
Stefanie
S
Köstner
Martin
M
Bertram
Ralph
R
Hillen
Wolfgang
W
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D003062:Codon / Q000235:genetics; D004351:Drug Resistance; D004926:Escherichia coli / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D003062:Codon; D011994:Recombinant Proteins; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein