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Characterization of the sequence‐specific interaction of mouse c‐myb protein with DNA.
Author(s) -
Howe K.M.,
Reakes C.F.,
Watson R.J.
Publication year - 1990
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1002/j.1460-2075.1990.tb08092.x
Subject(s) - biology , myb , dna , tandem repeat , helix turn helix , dna binding protein , microbiology and biotechnology , biochemistry , peptide sequence , electrophoretic mobility shift assay , amino acid , coding region , genetics , transcription factor , gene , genome
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.