Searching for the Code of Ideal Protein-DNA-Recognition
A system is described which allows testing of specific protein-DNA-interactions. The system consists of two mutually compatible plasmids carrying different origins of replication and resistance markers. One plasmid carries a lac I gene in which the DNA-recognizing domain has been replaced by synthetic DNA saturated with restriction sites. The other carries a lac P Z unit in which the natural operator has been deleted and replaced by a unique restriction site. Into this restriction site any operator can be cloned.
Our results suggest that: 1) The innermost seven base pairs of the lac operator can not be replaced without diminishing repression. 2) If Tyrl G1N2 of the recognition helix of lac repressor is substituted by Vall Ala2, the mutant repressor recognizes the mutant operator TGTAAGC GCTTACA better than the ideal or any other lac operator variant. Finally, a model is presented which may describe the binding of an alpha helix to the deep groove of B-DNA.
KeywordsUnique Restriction Site Recognition Helix Catabolite Activator Protein Lambda Operator Catabolite Gene Activator Protein
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