Broad binding-site specificity and affinity properties of octamer 1 and brain octamer-binding proteins.
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.
Bendall
A J
AJ
Commonwealth Scientific and Industrial Research Organisation, Division of Biomolecular Engineering, Sydney Laboratory, North Ryde, New South Wales, Australia.
Sturm
R A
RA
Danoy
P A
PA
Molloy
P L
PL
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006367:HeLa Cells; D051863:Host Cell Factor C1; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D050811:Octamer Transcription Factor-1; D017421:Sequence Analysis; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; C497692:HCFC1 protein, human; C497693:Hcfc1 protein, mouse; D051863:Host Cell Factor C1; D050811:Octamer Transcription Factor-1; C494165:POU2F1 protein, human; C494166:Pou2f1 protein, mouse; D014157:Transcription Factors; D004247:DNA