N-TERMINAL DNA-BINDING DOMAINS CONTRIBUTE TO DIFFERENTIAL DNA-BINDING SPECIFICITIES OF NF-KAPPA-B P50 AND P65

We previously reported that either oxidation or alkylation of NF-kappaB in vitro abrogates DNA binding. We used this phenomenon to help elucidate structural determinants of NF-kappaB binding. We now demonstrate that Cys-62 of NF-kappaB p50 mediates the redox effect and lies within an N-terminal region required for DNA binding but not for dimerization. Several point mutations in this region confer a transdominant negative binding phenotype to p50. The region is highly conserved in all Rel family proteins, and we have determined that it is also critical for DNA binding of NF-kappaB p65. Replacement of the N-terminal region of p65 with the corresponding region from p50 changes its DNA-binding specificity towards that of p50. These data suggest that the N-terminal regions of p50 and p65 are critical for DNA binding and help determine the DNA-binding specificities of p50 and p65. We have defined within the N-terminal region a sequence motif, R(F/G)(R/K)YXCE, which is present in Rel family proteins and also in zinc finger proteins capable of binding to kappaB sites. The potential significance of this finding is discussed.