NMR studies of the Escherichia coli Trp repressor.trpRs operator complex.
To understand the specificity of the Escherichia coli Trp repressor for its operators, we have begun to study complexes of the protein with alternative DNA sequences, using 1H-NMR spectroscopy. We report here the 1H-NMR chemical shifts of a 20-bp oligodeoxynucleotide containing the sequence of a symmetrised form of the trpR operator in the presence and absence of the holorepressor. Deuterated protein was used to assign the spectrum of the oligodeoxynucleotide in a 37-kDa complex with the Trp holorepressor. Many of the resonances of the DNA shift on binding to the protein, which suggests changes in conformation throughout the sequence. The largest changes in shifts for the aromatic protons in the major groove are for A15 and G16, which are thought to hydrogen bond to the protein, possibly via water molecules. We have also examined the effect of DNA binding on the corepressor, tryptophan, in this complex. The indole proton resonance of the tryptophan undergoes a downfield shift of 1.2 ppm upon binding of DNA. This large shift is consistent with hydrogen bonding of the tryptophan to the phosphate backbone of the trpR operator DNA, as in the crystal structure of the holoprotein with the trp operator.
Evans
P D
PD
School of Biochemistry, University of Birmingham, Edgbaston, UK.
Jaseja
M
M
Jeeves
M
M
Hyde
E I
EI
D001426:Bacterial Proteins / Q000737:chemistry; D004268:DNA-Binding Proteins; D003852:Deoxyribonucleoproteins / Q000737:chemistry; D004926:Escherichia coli; D006860:Hydrogen Bonding; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D009875:Operator Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D003852:Deoxyribonucleoproteins; D046911:Macromolecular Substances; D012097:Repressor Proteins; D014364:Tryptophan; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins