The role of the N terminus in Tet repressor for tet operator binding determined by a mutational analysis.
The N-terminal residues preceding the alpha-helix-turn-alpha-helix motif on the Tn10 Tet repressor protein were probed by oligonucleotide-directed deletion mutagenesis for their role in protein activity. All deletion mutants showed decreased repression in vivo, emphasizing the importance of the N terminus for tet operator binding. Only two of the mutants, TetR delta 2-23 and TetR delta 3-8 displayed a reduced intracellular protein level. The remaining deletion mutants showed either reduced binding to tet operator and inducibility by tetracycline or transdominance. We conclude that these deletions do not affect stability and overall protein structure. DNA binding activities of residue-wise increasing deletions, TetR delta 9 through TetR delta 9-13, reveal a pattern consistent with an alpha-helical structure of the affected residues. This conclusion is supported by the helical wheel projection and the hydrophobic moment profile calculated for the protein segment ranging from residues S7-V20. We propose that these residues form an amphipathic alpha-helix which packs closely against the alpha-helix-turn-alpha-helix motif and is essential for Tet repressor activity. The residues preceding this putative alpha-helix contribute to DNA binding, but no direct interactions with base pairs of tet operator were revealed in a loss of contact analysis. Individual mutation of the 4 charged residues to alanine at the N terminus shows that no single residue can account for the reduction in repression observed for the deletion mutants.
Berens
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C
Lehrstuhl für Mikrobiologie, Friedrich-Alexander Universität Erlangen-Nürnberg, Federal Republic of Germany.
Altschmied
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Hillen
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D000483:Alleles; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D002872:Chromosome Deletion; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009876:Operon; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D011994:Recombinant Proteins; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D001616:beta-Galactosidase