DNA sequence-specific recognition by a transcriptional regulator requires indirect readout of A-tracts.
The bacteriophage Ø29 transcriptional regulator p4 binds to promoters of different intrinsic activities. The p4-DNA complex contains two identical protomers that make similar interactions with the target sequence 5'-AACTTTTT-15 bp-AAAATGTT-3'. To define how the various elements in the target sequence contribute to p4's affinity, we studied p4 binding to a series of mutated binding sites. The binding specificity depends critically on base pairs of the target sequence through both direct as well as indirect readout. There is only one specific contact between a base and an amino acid residue; other contacts take place with the phosphate backbone. Alteration of direct amino acid-base contacts, or mutation of non-contacted A.T base pairs at A-tracts abolished binding. We generated three 5 ns molecular dynamics (MD) simulations to investigate the basis for the p4-DNA complex specificity. Recognition is controlled by the protein and depends on DNA dynamic properties. MD results on protein-DNA contacts and the divergence of p4 affinity to modified binding sites reveal an inherent asymmetry, which is required for p4-specific binding and may be crucial for transcription regulation.
Mendieta
Jesús
J
Instituto de Biología Molecular Eladio Viñuela (CSIC), Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain.
Pérez-Lago
Laura
L
Salas
Margarita
M
Camacho
Ana
A
D017103:Bacillus Phages / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D003198:Computer Simulation; D004279:DNA, Viral / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012091:Repetitive Sequences, Nucleic Acid; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism
D004279:DNA, Viral; D014157:Transcription Factors; D014764:Viral Proteins; C053987:p4 protein, Bacteriophage phi 29