Intragenic complementation between Escherichia coli trp repressors with different defects in the tryptophan-binding pocket.
Site-directed mutagenesis of the trpR gene (encoding the trp repressor, TrpR) was used to replace Gly85 with tryptophan (Trp or W), in order to place Trp near its normal location in the L-tryptophan(L-W)-binding pocket. The resulting mutant protein (G85W) exhibits weak, but significant repressor activity in vivo that is independent of the presence of L-W in the media. This mutant negatively complements the chromosomal wild type (wt), but does not negatively complement either the wt or the super-repressor, E49K, when any of these alleles is expressed on a multicopy plasmid. Activity of the mutant repressor, G85W, when produced in vivo together with T44M, approaches that of the wt repressor. This result presumably reflects complementation between the two mutant polypeptides. Similar results are obtained when G85R or G85K are combined with T44M in vivo, but not when G85W is replaced by G85E. The level of repression is dependent on the presence of L-W in the media. The TrpR with two mutations altering both Gly85 (G85W, G85R, G85E or G85K) and Thr44 (T44M) has no repressor activity. These results suggest a type of site-specific intragenic complementation where only certain alterations at Gly85 complement T44M. In this study, a positive charge or an indole ring appears to be required for the observed intragenic complementation.
Storbakk
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Department of Biotechnology, University of Tromsø, Norway.
Oxender
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DL
el-Gewely
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MR
D000483:Alleles; D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics*; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009154:Mutation; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism*; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012097:Repressor Proteins; D014364:Tryptophan; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins