Construction of an artificial tandem protein of the c-Myb DNA-binding domain and analysis of its DNA binding specificity.
An artificial tandem protein was generated using the third repeat of the c-Myb DNA-binding domain, and its DNA binding affinity and specificity were analyzed by a filter binding assay, isothermal titration calorimetry, and surface plasmon resonance. Although this artificial protein had the proper secondary structure, which is similar to the third repeat by itself, it could not bind to the expected base sequences specifically. Compared with the successful results of the zinc finger fusion proteins with novel sequence specificities, the cooperativity between the adjacent repeats, observed in the c-Myb-DNA complex, should also be required for the DNA recognition by the artificial tandem protein. Using the previous analyses of the DNA binding specificities by Myb homologous proteins, the differences in the DNA recognition mechanisms between the animal and plant Myb domains are also discussed.
Oda
M
M
Biomolecular Engineering Research Institute (BERI), Furuedai, Suita, Osaka, 565-0874, Japan. oda@rs.noda.sut.ac.jp
Furukawa
K
K
Sarai
A
A
Nakamura
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002151:Calorimetry; D002942:Circular Dichroism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005998:Glycine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020598:Proto-Oncogene Proteins c-myb; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D020349:Surface Plasmon Resonance; D020080:Tandem Repeat Sequences / Q000235:genetics*; D013816:Thermodynamics; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D020598:Proto-Oncogene Proteins c-myb; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D004247:DNA; D005998:Glycine