Abstract
The Tn10-encoded Tet repressor contains two tryptophan residues at positions 43 and 75. The typical tryptophan fluorescence is decreased upon binding of tet operator. The Tet repressor gene was engineered to replace either or both of the Trp codons by Phe codons. The resulting single tryptophan mutants are called F43 and F75 and the double mutant F43F75. The mutant proteins were purified to homogeneity. They recognize tet operator DNA only in the absence of the inducer tetracycline, indicating an intact tertiary structure of the engineered proteins. F75 and wild-type bind tet operator with the same association constant. The association constants of F43 and F43F75 with tet operator are about 3 orders of magnitude smaller. This indicates that Trp43 is important for tet operator recognition. Trp43 fluorescence is completely quenched in the complex with tet operator DNA while Trp75 remains unaffected. Binding to nonspecific DNA leads only to a 40% decrease of Trp43 fluorescence. This is interpreted as the contribution of the changed environment while the complete quench reflects a tight sequence-specific contact of tryptophan 43 to tet operator DNA. Trp43 is solvent-exposed, while Trp75 is buried in the hydrophobic interior of the protein. These results are discussed in light of the alpha-helix turn-alpha-helix DNA binding motif deduced from homology to other repressor proteins.
Highlights
Evidence for protein-DNA contacts is obtained from alkylation protection or interference experiments of the recsmaller
TheseA sequence-specific contact between Trp43 and tet operator resultsare discussed in ligohftthe a-helix turn-a-helix DNA is established by fluorescence spectroscopy and quanti
We conclude that the wild type and F75 Tet repressors have identical equilibrium association constants with tet operator
Summary
From the Lehrstuhl fur Mikrobwlo-gie der Friedrich-Alerander-Uniuersit~Erlangen-Nurnberg, Staudtstr. 5,852 Erlangen, Federal Republic of Germuny. Inthis model sequence specificity is Themutantproteins were purified to homogeneity They recognize tet operator DNA only in the absence of the inducer tetracycline, indicating an intact tertiary structure of the engineered proteins. The molecular mation of Escherichia coli [14], preparation of plasmid DNA [15], basis of sequence specificity in procaryotic DNA binding proteins has been elucidated by x-ray analyses of the crystal structures of Trp repressor [4],cro repressor [5],the catabolite and gel electrophoreses on polyacrylamide and agarose [16, 17] were done exactly as described earlier. Turn-a-helix structural motif of the respective protein makes 5’-End labeling of the oligonucleotidesand DNA fragments with 3*P sequence-specificcontacts in the major groove of B-DNA [1] This model gained support when the recognition helix of the was done as published [20]. The annealing of the mutagenic oligonucleotides and the polymerase and ligase reac-
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