The missing nucleoside experiment: a new technique to study recognition of DNA by protein

Abstract

We report a new technique for quickly determining which nucleosides in a DNA molecule are contacted by a sequence-specific DNA-binding protein. Our method is related to the recently reported "missing contact" experiment [Brunelle, A., & Schleif, R. F. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 6673-6679]. We treat the DNA molecule with the hydroxyl radical to randomly remove nucleosides. The ability of protein to bind to gapped DNA is assayed by gel mobility shift. Nucleosides important to protein binding are identified by sequencing gel electrophoresis. The missing nucleoside experiment can be used to scan a DNA molecule at single-nucleotide resolution in one experiment. The bacteriophage lambda repressor-OR1 and cro-OR1 complexes were analyzed to evaluate the method. For both proteins, the most important contacts are located in the protein monomer that binds to the consensus half of the operator. These contacts correspond well to those found by mutational studies, and in the cocrystal structure of the lambda repressor-operator. The missing nucleoside data show that the amino-terminal arms of lambda repressor make energetically important contacts with positions 7 and 8 and the central dyad base pair of the operator. The amino-terminal arm that makes the most extensive contacts to DNA appears to be the one that emanates from the repressor monomer that binds to the consensus half of the operator, in agreement with the cocrystal structure. The lambda cro protein does not have an amino-terminal arm, and the missing nucleoside experiment clearly shows a lack of contacts to DNA in the central region of the operator in this complex.