DNA-BINDING SPECIFICITY OF NGFI-A AND RELATED ZINC-FINGER TRANSCRIPTION FACTORS

NGFI-A is the prototypic member of a family of immediate-early gene-en coded transcription factors which includes NGFI-C, Egr3, and Krox20. T hese proteins possess highly homologous DNA-binding domains, composed of three Cys(2)-His(2) zinc fingers, and all bind to and activate tran scription from the sequence GCGGGGGCG. We used a PCR-mediated random s ite selection protocol to determine whether other sites could be bound by these proteins and the extent to which their binding site preferen ces are similar or different. The high-affinity consensus sites genera ted from the selection data are similar, and the combined consensus se quence is T-G-C-G-T/g-G/A-G-G-C/a/t-G-G/T (lowercase letters indicate bases selected less frequently). Using gel shift assays, we found that sequences that diverge from the consensus were bound by NGFI-A, confi rming that there is greater variability in binding sites than has gene rally been acknowledged. We also provide evidence that protein-DNA int eractions not noted, or whose importance was not apparent from the X-r ay cocrystal structure of the NGFI-A zinc fingers complexed with DNA, contribute significantly to the binding energy of these proteins and c onfirm that an optimal site is at least 10 instead of 9 nucleotides in length. In contrast to the similarities in binding specificity among these proteins, we found that while NGFI-A, Egr3, and Krox20 have comp arable DNA binding affinities and kinetics of dissociation, the affini ty of NGFI-C is more than threefold lower. This could result in differ ential regulation of target genes in cells where NGFI-C and the other proteins are coexpressed. Furthermore, we show that this affinity diff erence is a property not of the zinc fingers themselves but rather of the protein context of the DNA-binding domain.