Broad binding-site specificity and affinity properties of octamer 1 and brain octamer-binding proteins.

Abstract

The ubiquitous Pit-1-Oct-1-Unc-1 (POU)-domain protein octamer 1 (Oct-1) has been observed to bind specifically to a number of degenerate and dissimilar sequences. We have used antibodies directed against a C-terminal Oct-1 peptide to immunoselect binding sequences for HeLa cell Oct-1 from random-sequence oligonucleotides and we describe the isolation of binding sequences of considerable heterogeneity. Although our consensus alignment indicated a 9-bp TATGCAAAT motif with AT-rich flanking sequences, this binding motif is not immediately obvious in the population of sequences and no clone actually contained this sequence. Screening these Oct-1-binding sequences with a mouse whole-brain extract demonstrated that the neuronal octamer-binding proteins exhibit similar but distinct DNA sequence specificities. Unlike the reported selection of binding sequences for other transcription factors, the dependence of Oct-1-binding affinity upon sequence did not correspond tightly to the degree of conservation at particular positions of the consensus sequence. Our results suggest that either base-specific hydrogen bonding is not the only major determinant of binding affinity and specificity, or that Oct-1 binding to some sequences is mechanistically different from its binding to an octamer. These results exemplify the potential to overlook binding sites for some factors by searching gene sequences with a consensus nucleotide sequence.