1H-NMR investigation of the interaction of the amino terminal domain of the LexA repressor with a synthetic half-operator.
A synthetic half-operator DNA-duplex, d(GCTACTGTATGT), containing a portion of the proposed recognition sequence (CTGT) of several "SOS" genes, has been synthesized. The dodecamer has been characterized through 1H-NMR spectroscopy. Complete assignment of exchangeable hydrogen bonded imino protons has been achieved by applying 1D NOE techniques and an analysis of the temperature dependence of the chemical shifts. In order to determine the specific role of the CTGT consensus sequence in the overall recognition process, the oligonucleotide duplex has been titrated with the amino terminal DNA binding domain of the LexA repressor. The observation of substantial changes of 1H-NMR chemical shifts in the imino proton region upon interaction with the protein strongly suggests that the protein binds specifically to the operator DNA. The largest deviations of 1H-NMR chemical shifts upon protein binding have been observed for protons assigned to the CTGT segment, thus strongly suggesting a direct involvement of this sequence in the binding process. At high potassium chloride concentrations the 1H-NMR chemical shift deviations are reverted which is consistent with the known drop in the affinity constant of LexA for operator DNA at high salt concentrations.
Ottleben
G
G
Institute of Biophysical Chemistry, Johann Wolfgang Goethe-University of Frankfurt, Germany.
Messori
L
L
Rüterjans
H
H
Kaptein
R
R
Granger-Schnarr
M
M
Schnarr
M
M
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D004926:Escherichia coli; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D004247:DNA; D003854:Deoxyribonucleotides; D012097:Repressor Proteins; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein