INTERACTION OF TET REPRESSOR WITH OPERATOR DNA AND WITH TETRACYCLINE STUDIED BY INFRARED AND RAMAN-SPECTROSCOPY

Tet repressor (TetR) is involved in the most abundant mechanism of tet racycline (Tc) resistance of Gramnegative bacteria. Raman spectra were measured for the class D TetR protein, for an oligodeoxyribonucleotid e with sequence corresponding to operator site O1, and for the TetR:ol igonucleotide complex. TetR forms a complex with [Ni-Tc](+), which doe s not bind to operator DNA. Raman and infrared measurements indicate n early identical conformations of TetR with and without [Ni-Tc](+). Dif ferences between the experimental spectrum of the TetR:operator DNA co mplex and the computed sum of the component spectra provide direct spe ctroscopic evidence for changes in DNA backbone torsions and base stac king, rearrangement of protein backbone, and specific contacts between TetR residues and DNA bases. Complex formation is connected with inte nsity decrease at 1376 cm(-1) (participation of thymine methyl groups) , intensity increase at 1467 cm(-1) (hydrogen bond formation at guanin e N7), decreased intensity ratio i(854)/i(823) (increased hydrophobici ty of tyrosine environment), increased intensity at 1363 cm(-1) (incre ased hydrophobicity of tryptophan ring environment), differences in th e range 670-833 cm(-1) (changes in B-DNA backbone torsions and base st acking), and decreased intensity of the amide I band (structural rearr angement of TetR backbone consistent with a reduction of the distance between the two binding helices).