Raman spectroscopic analysis of Tet repressor-operator DNA interaction in deuterium oxide

Tet repressor (TetR) plays a central role in the regulation of its own gene and in that of TetA, a resistance protein against the antibiotic tetracycline (Tc). In the absence of Tc, the TetR dimer binds with two alpha-helix-turn-alpha-helix motifs to two successive major grooves of operator DNA. In order to elucidate structural features of the TetR:operator complex, we measured the Raman spectra of the TetR protein, a 18-mer oligonucleotide with sequence corresponding to TetR operator DNA, and the TetR:operator complex in D2O. The spectra confirm and extend previously obtained results in H2O: i) B-DNA conformation is conserved with only small perturbations of the backbone geometry; ii) TetR and operator DNA interact at major groove sites, as evident from intensity changes of thymine and guanine bands; iii) Minor changes of TetR secondary structure are indicated upon operator binding, and iv) Local environments of aromatic amino acids are altered in the complex. These spectroscopic findings are consistent with a molecular model proposed of the basis of genetic and biochemical studies.