ACQUISITION OF NFKB1-SELECTIVE DNA-BINDING BY SUBSTITUTION OF 4 AMINO-ACID-RESIDUES FROM NFKB1 INTO RELA

The subunits of NF-kappaB, NFKB1 (formerly p50) and RelA (formerly p65 ), belong to a growing family of transcription factors that share exte nsive similarity to the c-rel proto-oncogene product. The homology ext ends over a highly conserved stretch of approximately 300 amino acids termed the Rel homology domain (RHD). This region has been shown to be involved in both multimerization (homo- and heterodimerization) and D NA binding. It is now generally accepted that homodimers of either sub unit are capable of binding DNA that contains a KB site originally ide ntified in the immunoglobulin enhancer. Recent studies have demonstrat ed that the individual subunits of the NF-kappaB transcription factor complex can be distinguished by their ability to bind distinct DNA seq uence motifs. By using NFKB1 and RelA subunit fusion proteins, differe nt regions within the RHD were found to confer DNA-binding and multime rization functions. A fusion protein that contains 34 N-terminal amino acids of NFKB1 and 264 amino acids of RetA displayed preferential bin ding to an NFKB1-selective DNA motif while dimerizing with the charact eristics of RelA. Within the NFKB1 portion of this fusion protein, a s ingle amino acid change of His to Arg altered the DNA-binding specific ity to favor interaction with the RetA-selective DNA motif. Furthermor e, substitution of four amino acids from NFKB1 into RetA was able to a lter the DNA-binding specificity of the RetA protein to favor interact ion with the NFKB1-selective site. Taken together, these findings demo nstrate the presence of a distinct subdomain within the RHD involved i n conferring the DNA-binding specificity of the Rel family of proteins .