Abstract
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.
Highlights
THEJOURNALOF BIOLOGICAL CHEMISTRY0 1992 by The American Society for Biochemistry and Molecular Biology, Inc. VOl. 267, No., Issue of January 25, pp. 1945-1952,1992 Printed in U.S.A
The Role of Terminus in Tet Repressor for tet Operator Binding Determined bya Mutational Analysis*
The N-terminal residues preceding thae-helix-turn- bonds or van der Waals contacts with the edges of the base a-helix motif of the TnlO Tet repressor protein were pairs
Summary
0 1992 by The American Society for Biochemistry and Molecular Biology, Inc. VOl. 267, No., Issue of January 25, pp. 1945-1952,1992 Printed in U.S.A. Level of transcription initiation by proteins that bind to spe- no functional role has been attributed to thefirst 25 residues cific DNA sequences and act as repressors or activators. The plasmid pWHlO12 andits operator synthesized by the phosphoramidate method (Matteucci and Caruth- mutant derivatives express different levels of @-galactosidasein the ers, 1981) on an Applied Biosystems DNA synthesizer model 381A absence of Tet repressor (Sizemore et al, 1990). Determination of the Steady-state Levels of Wild Type and Mutant Tet Repressor Proteins-An overnight culture of E. coli WH207 transformed with pWHlO12 and pWH1201, pWH520, or a pWH520 derivative was diluted 100-fold in 40 ml of fresh LB supplemented with ampicillin (100 mg/liter) and kanamycin (60 mg/liter) and incubated a t 22, 28, or 37 "C. Mutants were identified by sequencing(Sangeret aL, 1977)and yieldswere between 4 (tetRA223) and 75% (tetRA9)
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