Asymmetric DNA binding by a homodimeric bHLH protein.
Protein-DNA interactions that lie outside of the core recognition sequence for the Drosophila bHLH transcription factor Deadpan (Dpn) were investigated using minor groove binding pyrrole-imidazole polyamides. Electrophoretic mobility shift assays and DNase I footprinting demonstrate that hairpin polyamides bound immediately upstream, but not immediately downstream of the Dpn homodimer selectively inhibit protein-DNA complex formation. Mutation of the Dpn consensus binding site from the asymmetric sequence 5'-CACGCG-3' to the palindromic sequence 5'-CACGTG-3' abolishes asymmetric inhibition. A Dpn mutant containing the unnatural amino acid norleucine in place of lysine at position 80 in the bHLH loop region is not inhibited by the polyamide, suggesting that the epsilon amino group at this position is responsible for DNA contacts outside the major groove. We conclude that the nonpalindromic Dpn recognition site imparts binding asymmetry by providing unique contacts to the basic region of each monomer in the bHLH homodimer.
Winston
R L
RL
Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Ehley
J A
JA
Baird
E E
EE
Dervan
P B
PB
Gottesfeld
J M
JM
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D003850:Deoxyribonuclease I / Q000737:chemistry; D019281:Dimerization; D004330:Drosophila; D029721:Drosophila Proteins; D018257:Helix-Loop-Helix Motifs / Q000235:genetics; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D009757:Nylons / Q000737:chemistry; D015345:Oligonucleotide Probes / Q000138:chemical synthesis; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D011485:Protein Binding / Q000235:genetics
D051792:Basic Helix-Loop-Helix Proteins; D004247:DNA; D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I; D029721:Drosophila Proteins; D008239:Lysine; D009687:Nuclear Proteins; D009757:Nylons; D015345:Oligonucleotide Probes; D010446:Peptide Fragments; C078020:Dpn protein, Drosophila