Bacterial expression, characterization and DNA binding studies on Drosophila melanogaster c-Myb DNA-binding protein.
The Drosophila Myb homologue retains an evolutionarily conserved typical sequence of three imperfect tandem tryptophan repeat units (R1-R2-R3) of 51-53 amino acids towards its N-terminus as its presumptive DNA binding domain. Using PCR amplification and the T7 expression vector pET 11d, we have overproduced this tryptophan repeat domain of Drosophila Myb in Escherichia coli and the protein has been purified. Circular dichroic measurements indicate that the protein has a high helical component (58.6%) in its overall structure. The protein is found to recognize the same cognate target sequence TAACGG, as recognized by the vertebrate proteins. The DNA binding properties of the protein have been investigated in detail by fluorescence spectroscopy taking advantage of the large number of tryptophan residues present in the protein. The fluorescence of the native Drosophila R123 was quenched when synthetic duplex DNA oligomers were added to the protein. The oligomers containing specific Myb target sites quenched the protein fluorescence to a greater extent than the non-specific DNA. Binding constants of the protein to the targets were also length dependent for smaller oligomers. Experiments with the collisional quencher acrylamide and cysteine modification reagent indicated that the specific and non-specific target sequences interact with the protein differently. In the former case both the buried and the exposed tryptophan residues were affected by DNA binding whereas in the latter only the solvent-exposed residues were involved.
Madan
A
A
Tata Institute of Fundamental Research, Bombay, India.
Radha
P K
PK
Hosur
R V
RV
Padhy
L C
LC
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020598:Proto-Oncogene Proteins c-myb
D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D020598:Proto-Oncogene Proteins c-myb; D004247:DNA