A simple screening for mutant DNA binding proteins: application to murine transcription factor PEBP2alpha subunit, a founding member of the Runt domain protein family.
Mouse transcription factor PEBP2 (polyomavirus enhancer-binding protein (2) is composed of two distinct subunits alpha and beta. The alpha subunit has an ability to bind the specific DNA sequences, which is enhanced by formation of a heterodimer with the beta subunit. The DNA binding and heterodimerization activities of the alpha subunit are both localized within a 128-amino-acid (aa) region termed as the Runt domain for its homology to the Drosophila segmentation gene runt. To characterize the molecular determinants for these activities, the Runt domain was randomly mutagenized and produced in E. coli as a secreted form. Using E. coli culture supernatant, the DNA binding and heterodimerization of mutant Runt domains were analyzed by gel retardation assay. Nine randomly picked single-aa substitution mutants showed various functional alterations in DNA binding and heterodimerization either separately or simultaneously. This observation suggests that the structure of Runt domain is highly ordered and is quite sensitive to modulations in its primary structure. The method presented here provides a simple and quick method to characterize a large number of mutant DNA binding proteins.
Akamatsu
Y
Y
Department of Genetics and Molecular Biology, Institute for Virus Research, Kyoto University, Syogoin, Sakyo-ku, Japan.
Tsukumo
S
S
Kagoshima
H
H
Tsurushita
N
N
Shigesada
K
K
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D007763:Lac Operon; D051379:Mice; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D016415:Sequence Alignment; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology
D004268:DNA-Binding Proteins; D050656:Transcription Factor AP-2; D014157:Transcription Factors