Conformational changes induced by DNA binding of NF-kappa B.
The transcription factor NF-kappa B makes extensive contacts with its recognition site over one complete turn of the double helix. Structural transitions, in both protein and DNA, that accompany formation of the DNA-protein complex were analysed by proteinase sensitivity and circular dichroism (CD) spectroscopy. In the absence of DNA chymotrypsin cleaved p50 after residues Y60 and N78, while proteinase K cleaved p50 after residues S74 and Q180. Previous experiments had indicated that trypsin cleaved p50 after K77. Cleavages after Y60, S74, K77 and N78 were blocked in the presence of bound DNA, whereas cleavage after Q180 was enhanced. Y60, S74, K77 and N78 are all located in the p50 N-terminal domain AB loop, whereas Q180 is located in the mainly alpha-helical region between p50 N-terminal domain beta-strands G' and H. As only Y60 makes direct contact with the DNA it is likely that the AB loop is highly unstructured in the absence of DNA, but is held in a rigid, proteinase-resistant structure by bound DNA. These conclusions were supported by CD spectroscopic studies of recombinant p50 and p65 homodimers, which indicated that both species changed conformation when binding DNA. Examination of the near UV CD spectra revealed that with some DNA sequences the bound and free forms of the DNA assumed different conformations. While this was evident for a fully symmetrical, high affinity recognition site DNA, it was not apparent with less tightly bound DNA.
Matthews
J R
JR
School of Biological and Medical Sciences, University of St Andrews, Fife, UK.
Nicholson
J
J
Jaffray
E
E
Kelly
S M
SM
Price
N C
NC
Hay
R T
RT
D001483:Base Sequence; D002918:Chymotrypsin; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D003852:Deoxyribonucleoproteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D017434:Protein Structure, Tertiary; D014357:Trypsin
D004268:DNA-Binding Proteins; D003852:Deoxyribonucleoproteins; D016328:NF-kappa B; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D004247:DNA; D002918:Chymotrypsin; D014357:Trypsin