The possible roles of residues 79 and 80 of the Trp repressor from Escherichia coli K-12 in trp operator recognition.
We constructed mutants of the Trp repressor from Escherichia coli K-12 with all possible single amino acid exchanges at positions 79 and 80 (residues 1 and 2 of the recognition helix). We tested these mutants in vivo by measuring the repression of synthesis of beta-galactosidase with symmetric variants of alpha- and beta-centered trp operators, which replace the lac operator in a synthetic lac system. The Trp repressor carrying a substitution of isoleucine 79 by lysine, showed a marked specificity change with respect to base pair 7 of the alpha-centered trp operator. Gel retardation experiments confirmed this result. Trp repressor mutant IR79 specifically recognizes a trp operator variant with substitutions in positions 7 and 8. Another mutant, with glycine in position 79, exhibited loss of contact at base pair 7. We speculate that the side chain of Ile79 interacts with the AT base pairs 7 and 8 of the alpha-centered trp operator, possibly with the methyl groups of thymines. Replacement of thymine in position 7 or 8 by uracil confirms the involvement of the methyl group of thymine 8 in repressor binding. Several Trp repressor mutants in position 80 (i.e. A180, AL80, AM80 and AP80) broaden the specificity of the Trp repressor for alpha-centered trp operator variants with exchanges in positions 3, 4 and 5.
Güneş
C
C
Institut für Genetik, Universität zu Köln, Germany.
Staacke
D
D
von Wilcken-Bergmann
B
B
Müller-Hill
B
B
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001482:Base Composition; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000378:metabolism*; D004794:Enzyme Repression; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D018257:Helix-Loop-Helix Motifs; D007763:Lac Operon / Q000235:genetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000096:biosynthesis; D001616:beta-Galactosidase / Q000235:genetics; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014364:Tryptophan; D001616:beta-Galactosidase; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins