IDEAL DNA BINDING SITE SPACING BY NF-KB P50 HOMODIMER

Includes bibliographical references (pages 29-30).NF-??B is a family of eukaryotic transcription factor proteins that inducibly regulates the expression of a large number of genes that play important roles in innate immunity and inflammation. NF-??B consists of varying homo- and heterodimers, one form of which is a homodimer of p50 subunits (p50:p50). The NF-??B p50 homodimer is unique among NF-??B proteins in that it escapes regulation through association with classical I??B proteins in the cytoplasm and enters the nucleus where it binds to DNA. In the nucleus, p50:p50 is the preferred binding partner of the ???nuclear I??B??? protein I??B??, itself an NF-??B responsive protein that is required for the further NF-??B-dependent expression of the pluripotent cytokine interleukin-6 (IL-6). This raises the possibility that ternary complexes of NF-??B p50 homodimer with I??B?? on target gene DNA could serve as a necessary signal for elevated expression of vital pro-inflammatory NF-??B target genes. Previously determined x-ray crystal structures of p50:p50 on ??B DNA have revealed that this dimeric transcription factor binds to double-stranded DNA with variable spacing. We recently crystallized and determined the x-ray crystal structure of the DNA binding Rel homology region of murine p50 homodimer to ??B DNA from the promoter of the IL-6 and NGAL genes and found, once again, that p50 binds the two sites with distinct spacing. In an attempt to identify the ???preferred??? binding mode of p50 on ??B DNA, we prepared oligonucleotides with an ???idealized??? sequence ??B DNA designed to allow p50:p50 to bind with 9-12 base pair spacing. The 3.0 ?? x-ray co-crystal structure of the complex suggests that p50:p50 binds ??B DNA with 11 base pair spacing when afforded freedom to select its ideal binding mode. We conclude that this is the preferred spacing of p50:p50 on ??B DNA