Homeodomain determinants of major groove recognition.
The homeodomain is a highly conserved structural module that binds DNA and participates in protein-protein interactions. Most homeodomains contain residues at positions 47 and 51 which mediate recognition of a TAAT core binding sequence in the major groove. The constraints imposed on the identity of these residues by homeodomain structure and DNA docking have been examined in the context of the POU domain of the Oct-1 transcription factor. A bacterial library, in which POU homeodomain residues 47 and 51 have been randomized, was probed on nitrocellulose filters for the binding of DNA fragments containing the consensus octamer sequence. The residues which provide for the highest affinity interaction with the octamer consensus sequence, and the greatest specificity, are the highly conserved wild-type residues valine 47 and asparagine 51. Interestingly, a class of variants containing arginine at position 51 was also detected in the screen and found to have moderate affinity for the consensus sequence but reduced specificity compared to the wild-type protein. A single variant containing arginine at both positions 47 and 51 was detected when the library was probed with fragments containing nucleotide substitutions at positions expected to be contacted by residues 47 and 51. This variant was used to alter the DNA-binding specificity of a transcriptional regulatory complex which depends upon Oct-1 for DNA recognition. These findings suggest that homeodomain structure and DNA docking constrain in the versatility of the domain in that only a limited set of amino acid determinants can endow the domain with specific, high-affinity DNA binding.
Pomerantz
J L
JL
Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139.
Sharp
P A
PA
D001483:Base Sequence; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D015723:Gene Library; D005801:Genes, Homeobox; D051863:Host Cell Factor C1; D008969:Molecular Sequence Data; D050811:Octamer Transcription Factor-1; D050809:POU Domain Factors; D011485:Protein Binding; D011487:Protein Conformation; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D051863:Host Cell Factor C1; D050811:Octamer Transcription Factor-1; D050809:POU Domain Factors; D014157:Transcription Factors