Figure 2

The mR3-3 is defective in transcriptional activation and this phenotype can be rescued by VP16 fusion to the mutant DNA-binding domain. A. Alanine mutagenesis of the third c-Myb repeat was performed to analyze the structural and functional contributions of the acidic patch in the first helix of each repeat. Three mutants of the third repeat (mR3-1,2,3) were constructed: mR3-1 preserved the salt bridge and converted two acidic residues to alanines, mR3-2 eliminated the salt bridge, and mR3-3 preserved the salt bridge and changed all three acidic residues not involved in salt bridges to alanines. B. The diagram identifies the positions of the DNA-binding domain (DBD), the transcriptional activation domain (TA), and the C-terminal negative regulatory domain. The DNA-binding domain consists of three imperfect repeats labeled R1, R2, and R3. The truncated form of c-Myb (c-MybΔ) is missing the negative regulatory domain. c-MybVP represents the c-Myb DBD fused to the VP16 activation domain. C. Vector only (v/o), wild type (wt) c-Myb, or the mR3-3 c-Myb mutant constructs in (A) were tested for transcriptional activation of the EW5-E1b-Luciferase reporter. Luciferase activity for each of the mutant c-Myb constructs was normalized for transfection efficiency by measuring β-galactosidase activity from a cotransfected CMV-β-galactasidase plasmid. The wild-type c-Myb values were then set to 100% for each class of proteins and the mutants were scaled accordingly, allowing the comparison of multiple experiments and comparison of c-Myb, c-MybΔ, and c-MybVP. Before normalization the wild-type c-MybΔ and c-MybVP had a transcriptional activity 100× and 1000× greater than the full length c-Myb, respectively.