Two functionally distinct half sites in the DNA-recognition sequence of the Myb oncoprotein.
The oncoprotein Myb is a sequence-specific DNA-binding protein with a pivotal function in the development and proliferation of hematopoietic precursor cells. A minimal DNA-binding domain composed of two tryptophan-rich repeats R2 and R3 is responsible for sequence recognition. Based on model building and mutational analysis, Myb was proposed to recognise its target through a double helix-turn-helix (HTH)-related motif using two recognition helices, one in R2 and one in R3. We found, by mutational analysis, that the DNA-binding site for c-Myb is functionally bipartite. While the first half site is dominant and absolutely required for binding, the second half site is only modulatory and mainly affects the half life of the complex. This bipartite nature of the binding site parallels the proposed bipartite structure of R2R3 with two HTH-related domains. Analysis of the DNA-binding site of R2R3 by missing-base interference-footprint analysis showed that the protein interacted with a 9-bp region. The same was found with a larger protein containing all three repeats. The effect of adding R1 was mainly to stabilise the complex. The borders of the complex, as revealed by exonuclease III footprinting, did not change due to the presence of R1. However, both borders became more refractory to the nuclease when R1 was present, but with a difference that suggested a specific orientation of the repeat domains relative to the DNA-binding site. We propose that the first half site is recognised by R3, while the second modulatory half site interacts with the R2 repeat.
Ording
E
E
Department of Biochemistry, University of Oslo, Norway.
Kvåvik
W
W
Bostad
A
A
Gabrielsen
O S
OS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002645:Chickens; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018257:Helix-Loop-Helix Motifs; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D020598:Proto-Oncogene Proteins c-myb; D011994:Recombinant Proteins / Q000378:metabolism
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011518:Proto-Oncogene Proteins; D020598:Proto-Oncogene Proteins c-myb; D011994:Recombinant Proteins; D004247:DNA