Characterization of the sequence-specific interaction of mouse c-myb protein with DNA.
We have examined parameters that affect sequence-specific interactions of the mouse c-myb protein with DNA oligomers containing the Myb-binding motif (CA/CGTTPu). Complexes formed between these oligomers and in vitro translated c-myb proteins were analysed by electrophoresis on non-denaturing polyacrylamide gels using the mobility-shift assay. By progressive truncation of c-myb coding sequences it was demonstrated that amino acids downstream of a region of three imperfect 51-52 residue repeats (designated R1, R2 and R3), which are located close to the amino terminus of the protein, had no qualitative or quantitative effect on the ability to interact specifically with this DNA motif. However, removal of only five amino acids of the R3 repeat completely abolished this activity. The contribution of individual DNA-binding domain repeats to this interaction was investigated by precisely deleting each individually: it was demonstrated that a combination of R2 and R3 was absolutely required for complex formation while the R1 repeat was completely dispensible. c-myb proteins showed quantitatively greater interaction with oligomers containing duplicated rather than single Myb-binding motif, in particular where these were arranged in tandem. Moreover, it was observed that c-myb protein interacted with these tandem motifs as a monomer. These findings imply that a single protein subunit straddles adjacent binding sites and the implications for c-myb activity are discussed.
Imperial Cancer Research Fund Laboratories, St Bartholomew's Hospital, London, UK.
K M
KM
Howe
C F
CF
Reakes
R J
RJ
Watson
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D020598:Proto-Oncogene Proteins c-myb; D012091:Repetitive Sequences, Nucleic Acid; D014158:Transcription, Genetic
D011518:Proto-Oncogene Proteins; D020598:Proto-Oncogene Proteins c-myb; D004247:DNA