title: Mutations that improve the binding of yeast FLP recombinase to its substrate.
abstract: When yeast FLP recombinase is expressed from the phage lambda PR 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.
authors.affiliation: Department of Biological Sciences, University of Southern California, Los Angeles 90089-1481.
authors.forename: B
authors.initials: B
authors.lastname: Lebreton
authors.forename: P V
authors.initials: PV
authors.lastname: Prasad
authors.forename: M
authors.initials: M
authors.lastname: Jayaram
authors.forename: P
authors.initials: P
authors.lastname: Youderian
mesh_terms: D003062:Codon; D004254:DNA Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D010957:Plasmids; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D012486:Salmonella typhimurium / Q000235:genetics
chemical_list: D003062:Codon; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase