A genetic method for defining DNA-binding domains: application to the nuclear receptor NGFI-B.
A method is described that allows for rapid and efficient generation of functional mutations in DNA-binding domains of proteins. The target DNA-binding domain is attached to the Gal4p transcriptional-activating domain and expressed in yeast. The binding site recognized by the target domain is placed upstream of a gene that produces a protein toxic to yeast cells, so that the chimeric protein activates its expression, providing a selection against DNA-binding domain function. The chimeric protein also activates expression of a gene necessary for histidine prototrophy, using a second DNA-binding domain included in the chimera (lexA), providing a selection against general activator mutations. Therefore, requiring growth in the absence of histidine focuses mutations to the target DNA-binding domain. This method was applied to the DNA-binding domain of the nuclear receptor NGFI-B. Nearly all mutations obtained concurred with previous studies of NGFI-B and other nuclear receptors, verifying the functional validity of the mutational profile obtained. In addition, by coupling this selection scheme with the two-hybrid system [Chien, C.-t., Bartel, P. L., Sternglanz, R. & Fields, S. (1991) Proc. Natl. Acad. Sci. USA 88, 9578-9582], mutations that alter protein interaction domains could also be obtained.
Wilson
T E
TE
Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110.
Padgett
K A
KA
Johnston
M
M
Milbrandt
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D001665:Binding Sites; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D005690:Galactose / Q000494:pharmacology; D005800:Genes, Fungal; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D017433:Protein Structure, Secondary; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases; D016385:TATA Box; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D005690:Galactose; D005982:Glutathione Transferase; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D014157:Transcription Factors; D000099160:LexA Repressor Protein