Genetics, Vol 118, 393-400, Copyright © 1988

INVESTIGATIONS

Mutations That Improve the Binding of Yeast FLP Recombinase to Its Substrate

B. Lebreton, P. V. Prasad, M. Jayaram and P. Youderian
Department of Biological Sciences, University of Southern California, Los Angeles, California 90089-1481

When yeast FLP recombinase is expressed from the phage {lambda} P(R) promoter in a Salmonella host, it cannot efficiently repress an operon controlled by an operator/promoter region that includes a synthetic, target FLP site. On the basis of this phenotype, we have identified four mutant FLP proteins that function as more efficient repressors of such an operon. At least two of these mutant FLP proteins bind better to the FLP site in vivo and in vitro. One mutant changes the presumed active site tyrosine residue of FLP protein to phenylalanine, is blocked in recombination, and binds the FLP site about five-fold better than the wild-type protein. A second mutant protein that functions as a more efficient repressor retains catalytic activity. We conclude that the eukaryotic yeast FLP recombinase, when expressed in a heterologous prokaryotic host, can function as a repressor, and that mutant FLP proteins that bind DNA more tightly may be selected as more efficient repressors.

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