Synthesis of an enzymatically active FLP recombinase in vitro: search for a DNA-binding domain.
We have used an in vitro transcription and translation system to synthesize an enzymatically active FLP protein. The FLP mRNA synthesized in vitro by SP6 polymerase is translated efficiently in a rabbit reticulocyte lysate to produce enzymatically active FLP. Using this system, we assessed the effect of deletions and tetrapeptide insertions on the ability of the respective variant proteins synthesized in vitro to bind to the FLP recognition target site and to carry out excisive recombination. Deletions of as few as six amino acids from either the carboxy- or amino-terminal region of FLP resulted in loss of binding activity. Likewise, insertions at amino acid positions 79, 203, and 286 abolished DNA-binding activity. On the other hand, a protein with an insertion at amino acid 364 retained significant DNA-binding activity but had no detectable recombination activity. Also, an insertion at amino acid 115 had no measurable effect on DNA binding, but recombination was reduced by 95%. In addition, an insertion at amino acid 411 had no effect on DNA binding and recombination. On the basis of these results, we conclude that this approach fails to define a discrete DNA-binding domain. The possible reasons for this result are discussed.
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Department of Medical Genetics, University of Toronto, Ontario, Canada.
Sadowski
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D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D014176:Protein Biosynthesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D014158:Transcription, Genetic
D001425:Bacterial Outer Membrane Proteins; D004247:DNA; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase