The DNA binding specificity of the bipartite POU domain and its subdomains.
The POU domain is a conserved DNA binding region of approximately 160 amino acids present in a family of eukaryotic transcription factors that play regulatory roles in development. The POU domain consists of two subdomains, the POU-specific (POUS) domain and a POU-type homeodomain (POUHD). We show here that, like the POUHD, the Oct-1 POUS domain can bind autonomously to DNA but with low affinity. DNA binding studies and in vitro binding site selection revealed that the POU subdomains each have a different sequence specificity. The binding consensus of the POUS domain [gAATAT(G/T)CA] and POUHD (RTAATNA) respectively overlap the 'left half' and right half' of the POU domain recognition sequence [a(a/t)TATGC(A/T) AAT(t/a)t]. In addition to the core sequence, which is very similar to the octamer motif (ATGCAAAT), the flanking bases make a significant contribution to the binding affinity of the POU domain. Interestingly, at some positions the sequence preferences of the isolated POU subdomains are distinct from those of the POU domain, suggesting that the POU domain binding site is more than a simple juxtaposition of the POUS and POUHD target sequences. In addition, analysis of the binding kinetics of the POU domain and POUHD indicates that the POUS domain enhances the binding affinity by reducing the dissociation rate. Our results show that the POU domain proteins have DNA binding properties distinct from those of classic homeodomain proteins. We suggest a model for the way in which an additional conserved domain adds further specificity to DNA recognition by homeodomain proteins.
Laboratory for Physiological Chemistry, University of Utrecht, The Netherlands.
C P
CP
Verrijzer
M J
MJ
Alkema
W W
WW
van Weperen
H C
HC
Van Leeuwen
M J
MJ
Strating
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van der Vliet
D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005801:Genes, Homeobox; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; D014157:Transcription Factors; D004247:DNA