Deletion mutagenesis of Tn 10 Tet repressor--localization of regions important for dimerization and inducibility in vivo.
The gene for the Tn 10 Tet repressor (TetR) was subjected to deletion mutagenesis. Screening for a transdominant operator-binding negative phenotype yielded 10 mutants with internal deletions. Three deletions extend from residue D5 to residues L41, W75, or Q76, respectively, and two contain deletions of the alpha-helix-turn-alpha-helix DNA-binding motif. Five deletions range from residue K84 to residues between R87 and K98. Since residues from the N-terminus up to position 98 are not necessary for dimerization, this must take place in the C-terminal half of the protein. Ability to dimerize was probed by introducing ochre nonsense codons (oc) at residues G138, H151, E159, I174, or K202. Koc202 shows wild-type in vivo operator-binding and inducibility by tetracycline indicating that the six C-terminal residues of TetR are not important for activity. Mutants with longer C-terminal truncations are inactive and not transdominant. They show reduced steady-state protein levels and are probably impaired in folding and degraded in vivo. Two mutants (delta151-166, delta164-166) with deletions in a region variable in primary structure and length among Tet repressors from different resistance determinants bind tet operator efficiently, but are not inducible by tetracycline. This result indicates that these residues are not important for dimer formation in the operator-binding form.
Berens
C
C
Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie, Biochemie und Genetik der Friedrich-Alexander Universität Erlangen-Nürnberg, Germany.
Pfleiderer
K
K
Helbl
V
V
Hillen
W
W
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D018389:Codon, Nonsense; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D017384:Sequence Deletion; D013752:Tetracycline / Q000494:pharmacology; D014169:Transformation, Bacterial; D001616:beta-Galactosidase / Q000378:metabolism
D001426:Bacterial Proteins; D018389:Codon, Nonsense; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D001616:beta-Galactosidase; D013752:Tetracycline