Determining residue-base interactions between AraC protein and araI DNA.
Depurination/depyrimidation binding-interference experiments (missing contact probing) identified specific candidate residue-base interactions lost by mutants of Escherichia coli L-arabinose operon regulatory protein, AraC, to one of its binding sites, araI. These candidates were then checked more rigorously by comparing the affinities of wild-type and alanine-substituted AraC protein to variants of araI with alterations in the candidate contacted positions. Residues 208 and 212 apparently contact DNA and support, but do not prove the existence of a helix-turn-helix structure in this region of AraC protein whereas contacts by mutants with alterations at positions 256, 257 and 261 which are within another potential helix-turn-helix region do not support the existence of such a structure there. The missing contacts displayed by three AraC mutants are found within two major groove regions of the DNA and are spaced 21 base-pairs apart in a pattern indicating a direct repeat orientation for the subunits of AraC.
Brunelle
A
A
Department of Biochemistry, Brandeis University, Waltham, MA 02254.
Schleif
R
R
D001665:Binding Sites; D004252:DNA Mutational Analysis; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000032:analysis*; D005796:Genes; D005809:Genes, Regulator; D005797:Genes, araC; D005838:Genotype; D009154:Mutation; D011487:Protein Conformation; D012045:Regulatory Sequences, Nucleic Acid
D015342:DNA Probes; D004268:DNA-Binding Proteins