Figure 4.

Mutation analysis of the GRE consensus sequence. (A) Sequences of oligonucleotides used for competitive EMSAs m1–m4 contain triple base substitutions in the wild-type (wt) GRE sequence, and m5–m10 contain double base substitutions. m11 was mutated just outside of GRE, and m12–m14 contains a single base mutation at the sixth position of the GRE. (B) 100-fold molar excess of unlabeled probes was added to the binding reaction as cold competitors. The wild-type and m11 oligonucleotides efficiently competed for GST-ΔBTB-GZF1 binding to ³²P-labeled GRE, whereas m1–m10 cold competitors did not compete at all. An arrow indicates the DNA–protein complex and an arrowhead indicates free probe. (C) All oligonucleotides with single base substitutions at the sixth position of the GRE (m12–m14) competed efficiently for GST-ΔBTB-GZF1 binding to the ³²P-labeled GRE.