Proximity probing of Tet repressor to tet operator by dimethylsulfate reveals protected and accessible functions for each recognized base-pair in the major groove.

We have tracked the path of Tet repressor across the major groove in the complex with tet operator. This was done by a methylation protection analysis of nine tet operator mutants containing replacements by a G residue of each nucleotide in base-pairs important for Tet repressor recognition. We demonstrated sequence-specific binding of Tet repressor to these operator mutants using DNA retardation assays and the protection of the wild-type +2G residue from methylation. Hydroxyl radical cleavage protection analysis of the Tet repressor-tet operator complexes indicated identical, or at least very similar, locations of the DNA reading head across the major groove of wild-type and mutant operator DNA. Methylation protection occurred at the G residues in positions +3, +4, -5 and -6, whereas the G residues in the respective opposite strands showed enhanced methylation. These results show that most amino acid side-chains of Tet repressor are in close proximity to only one base of each base-pair in the major groove of tet operator. The Tet repressor mutant PS39 gave a changed methylation protection pattern at base-pair four of tet operator indicating that the residue at this position can contact either base at this base-pair depending on the amino acid side-chain present. Tet repressor mutants QA38 and TA40 with a loss of specificity phenotype gave the same methylation protection profile as wild-type TetR confirming that this experiment scores proximity rather than chemical interaction. The excellent agreement of these results with those obtained in genetic analyses demonstrates that this method yields a high-resolution proximity pattern of Tet repressor with tet operator and that it may be generally applicable for the analysis of protein-DNA complexes.