A genetic method for defining DNA-binding domains: application to the nuclear receptor NGFI-B.
AUTOR(ES)
Wilson, T E
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=47527Documentos Relacionados
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