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.
Krafft
C
C
Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany.
Hinrichs
W
W
Orth
P
P
Saenger
W
W
Welfle
H
H
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002021:Buffers; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D017666:Deuterium Oxide; D019077:Helix-Turn-Helix Motifs; D006639:Histidine / Q000737:chemistry; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009875:Operator Regions, Genetic; D010455:Peptides / Q000737:chemistry; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism; D013059:Spectrum Analysis, Raman / Q000379:methods*; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry; D014867:Water
D001426:Bacterial Proteins; D002021:Buffers; D004268:DNA-Binding Proteins; D010455:Peptides; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D014867:Water; D014443:Tyrosine; D006639:Histidine; D014364:Tryptophan; D017666:Deuterium Oxide