TY - JOUR
T1 - A genetic method for defining DNA-binding domains
T2 - Application to the nuclear receptor NGFI-B
AU - Wilson, Thomas E.
AU - Padgett, Kerstien A.
AU - Johnston, Mark
AU - Milbrandt, Jeffrey
PY - 1993/10/1
Y1 - 1993/10/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0027515629&partnerID=8YFLogxK
U2 - 10.1073/pnas.90.19.9186
DO - 10.1073/pnas.90.19.9186
M3 - Article
C2 - 8415675
AN - SCOPUS:0027515629
SN - 0027-8424
VL - 90
SP - 9186
EP - 9190
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 19
ER -