PROXIMITY PROBING OF TET REPRESSOR TO TET OPERATOR BY DIMETHYLSULFATEREVEALS 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 t he complex with tet operator. This was done by a methylation protectio n 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 protec tion of the wild-type +2G residue from methylation. Hydroxyl radical c leavage protection analysis of the Tet repressor-tet operator complexe s 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 str ands showed enhanced methylation. These results show that most amino a cid side-chains of Tet repressor are in close proximity to only one ba se of each base-pair in the major groove of tet operator. The Tet repr essor mutant PS39 gave a changed methylation protection pattern at bas e-pair four of tet operator indicating that the residue at this positi on can contact either base at this base-pair depending on the amino ac id 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 ra ther than chemical interaction. The excellent agreement of these resul ts with those obtained in genetic analyses demonstrates that this meth od yields a high-resolution proximity pattern of Tet repressor with te t operator and that it may be generally applicable for the analysis of protein-DNA complexes.