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 tetracycline (Tc) resistance of gram-negative bacteria. Raman spectra were measured for the class D TetR protein, for an oligodeoxyribonucleotide with sequence corresponding to operator site O1, and for the TetR:oligonucleotide complex. TetR forms a complex with [Ni-Tc]+, which does not bind to operator DNA. Raman and infrared measurements indicate nearly identical conformations of TetR with and without [Ni-Tc]+. Differences between the experimental spectrum of the TetR:operator DNA complex and the computed sum of the component spectra provide direct spectroscopic evidence for changes in DNA backbone torsions and base stacking, rearrangement of protein backbone, and specific contacts between TetR residues and DNA bases. Complex formation is connected with intensity decrease at 1376 cm(-1) (participation of thymine methyl groups), intensity increase at 1467 cm(-1) (hydrogen bond formation at guanine N7), decreased intensity ratio I854/I823 (increased hydrophobicity of tyrosine environment), increased intensity at 1363 cm(-1) (increased hydrophobicity of tryptophan ring environment), differences in the range 670-833 cm(-1) (changes in B-DNA backbone torsions and base stacking), and decreased intensity of the amide I band (structural rearrangement of TetR backbone consistent with a reduction of the distance between the two binding helices).
Krafft
C
C
Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany.
Hinrichs
W
W
Orth
P
P
Saenger
W
W
Welfle
H
H
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D019077:Helix-Turn-Helix Motifs; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009875:Operator Regions, Genetic; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism; D013055:Spectrophotometry, Infrared; D017550:Spectroscopy, Fourier Transform Infrared; D013059:Spectrum Analysis, Raman; D013752:Tetracycline / Q000737:chemistry* / Q000378:metabolism
D011994:Recombinant Proteins; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D004247:DNA; D013752:Tetracycline