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Intragenic complementation between Escherichia coli trp repressors with different defects in the tryptophan-binding
N Storbakk1, D L Oxender, M R el-Gewely
1Department of Biotechnology, University of Tromsø, Norway.
Gene
|August 1, 1992
Summary
Site-directed mutagenesis revealed that specific alterations at Gly85 in the trp repressor (TrpR) can restore function when combined with a Thr44 mutation, suggesting a requirement for positive charge or an indole ring for intragenic complementation.
Area of Science:
- Molecular Biology
- Protein Engineering
- Genetics
Background:
- The trp repressor (TrpR) regulates tryptophan biosynthesis.
- Understanding TrpR function requires studying mutations affecting its L-tryptophan binding pocket.
Purpose of the Study:
- To investigate the effects of specific mutations at Gly85 within the trpR gene.
- To explore intragenic complementation between mutations at Gly85 and Thr44 in TrpR.
Main Methods:
- Site-directed mutagenesis was employed to create specific amino acid substitutions in the trpR gene.
- In vivo assays were used to assess the repressor activity of mutant TrpR proteins.
- Complementation studies were performed with various mutant alleles.
Main Results:
- A G85W mutant TrpR exhibited weak, L-tryptophan-independent repressor activity.
- Complementation between G85W and T44M mutants restored near wild-type repressor activity.
- Intragenic complementation at Gly85 required specific alterations (positive charge or indole ring) and was dependent on L-tryptophan presence.
Conclusions:
- Specific Gly85 substitutions can mediate site-specific intragenic complementation with the T44M mutation.
- The findings highlight the structural requirements for TrpR function and interaction within the binding pocket.