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Mechanism of Tet repressor induction by tetracyclines: length compensates for sequence in the alpha8-alpha9 loop
1Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie Biochemie und Genetik, Friedrich-Alexander Universität Erlangen-Nurnberg, Germany.
Journal of Molecular Biology
|August 15, 2001
Summary
The alpha8-alpha9 loop in Tet repressor (TetR) is crucial for tetracycline induction. Loop length and specific residues influence inducer binding and conformational changes necessary for TetR function.
Area of Science:
- Molecular Biology
- Protein Engineering
- Biochemistry
Background:
- Natural Tet repressor (TetR) proteins possess a variable loop between helices 8 and 9.
- This loop's sequence and length significantly impact TetR's regulatory function.
Purpose of the Study:
- To investigate the role of the TetR alpha8-alpha9 loop in tetracycline induction.
- To determine how loop length and specific amino acid residues affect TetR's response to inducers.
Main Methods:
- Deletion and stepwise alanine substitution of the TetR alpha8-alpha9 loop.
- Assessing repression and induction efficiencies with tetracycline and anhydrotetracycline.
- Measuring binding constants for anhydrotetracycline-TetR interactions.
Main Results:
- TetR mutants with deleted or shortened alanine loops lost tetracycline inducibility.
- Induction efficiencies increased with loop length, particularly with anhydrotetracycline.
- Specific conserved residues (Asp/Glu157, Arg158) were critical for enhanced induction.
- Anhydrotetracycline binding affinity improved with loop length up to ten residues.
Conclusions:
- The alpha8-alpha9 loop's length and sequence are essential for TetR induction.
- Minimal loop length is required for efficient inducer binding.
- The loop structurally participates in the conformational changes required for TetR induction.