CYSTEINE-50 OF THE POUH-DOMAIN DETERMINES THE RANGE OF TARGETS RECOGNIZED BY POU-PROTEINS

The best target of POU proteins (Oct-1, Oct-2) is an octamer sequence ATGCAAAT, POU proteins also recognize, with weaker affinity, the TAAT- like targets of another group of regulatory factors, the homeoproteins , Up to now, it has not been known why Cys50 of the POUH domain is abs olutely conserved in contrast to that in homeoproteins, To assess the importance of Cys50 in determining the binding specificity of POU prot eins, all possible amino acids were substituted for Cys at position 50 , and the resulting mutants were tested with probes containing octamer (ATGCAAATNN) or homospecific binding sites, Only the wild-type POU wa s shown to adequately discriminate between the octamer and homospecifi c sites, and the protein affinity was only slightly affected by the nu cleotide sequence flanking the octamer at the 3'-end, Any amino acid s ubstitution at position 50 resulted in the mutant protein binding effi ciently both to the octamer and the TAAT-like sequences, Moreover, in this case the 3'-flanking sequences influenced the binding to a much g reater extent.