DNA binding by an amino acid residue in the C-terminal half of the Rel homology region.
BACKGROUND
Members of the Rel family of transcription factors are important in regulating the inflammatory, acute phase, and immune responses in mammals. The structural basis for sequence-specific binding by Rel proteins is poorly understood, however. In the studies reported here, a new photoaffinity labeling procedure has been used to probe DNA contacts established by a Rel protein, the p50 homodimer of NF-kappa B.


RESULTS
Using a novel post-synthetic modification method, 8-azido-2'-deoxyadenosine (N3dA), a photoactive analog of 2'-deoxyadenosine, was introduced at a specific site within a consensus DNA binding site for the NF-kappa B p50 homodimer. Upon irradiation with ultraviolet light, the N3dA-substituted DNA was efficiently photocrosslinked to p50. The crosslinked amino acid of p50 was identified as K244, which lies in the carboxy-terminal half of the Rel homology region (RHR). Mutation of K244 exerts strong effects on DNA binding, confirming the importance of this residue for p50-DNA interactions.


CONCLUSIONS
We have used N3dA-containing DNA to identify a residue of NF-kappa B p50 that contacts DNA illustrating the value of this approach in studies of protein-DNA interactions. K244 appears not to contact a DNA base, but rather a phosphate moiety that lies nearby. The segment of protein sequence in which K244 resides has been implicated in dimerization. The results presented here suggest that the DNA-binding domain extends farther toward the carboxy-terminus than previously thought.
Liu
J
J
Department of Chemistry, Harvard University, Cambridge, MA 02138, USA.
Fan
Q R
QR
Sodeoka
M
M
Lane
W S
WS
Verdine
G L
GL
D000596:Amino Acids / Q000378:metabolism*; D003432:Cross-Linking Reagents; D004247:DNA / Q000235:genetics*; D009154:Mutation; D016328:NF-kappa B / Q000235:genetics; D019920:Photoaffinity Labels; D012689:Sequence Homology, Nucleic Acid
D000596:Amino Acids; D003432:Cross-Linking Reagents; D016328:NF-kappa B; D019920:Photoaffinity Labels; D004247:DNA