An invariant asparagine in the POU-specific homeodomain regulates the specificity of the Oct-2 POU motif.
The homeodomain defines a family of transcription factors broadly involved in the regulation of gene expression. DNA recognition, as observed in three representative complexes (Engrailed, Antennapedia, and MAT alpha 2), is mediated in the major groove by a helix-turn-helix (HTH) element and in the minor groove by an N-terminal arm. The three complexes share similar overall features, but they also exhibit significant differences in DNA interactions. Because these differences may distinguish the biological activities of different classes of homeodomains, we have investigated the contribution of the Oct-2 POU-specific homeodomain (POUHD) to the specificity of the bipartite POU motif. Comparative studies of variant protein-DNA complexes demonstrate the following. (i) Mutations in an invariant residue in the POUHD HTH (N347; residue 10 of the putative recognition alpha-helix) reduce octamer binding with the relaxation of specificity at one position (5'-ATGCAAAT). The inferred HTH side chain-base interaction, although not observed in the solution structure of the Antennapedia complex, is in accord with homologous contacts in the Engrailed and MAT alpha 2 cocrystal structures. (ii) Comparison of the DNA-binding properties of POU and POUHD demonstrates that POUs and POUHD independently regulate specificity at opposite ends of the DNA site (5'-TATGCAAAT). Both domains contact the two central bases (5'-TATGCAAAT) where coordinate binding of POUS in the major groove overrides the intrinsic specificity of POUHD in the minor groove. (iii) The differential sensitivity of POU and POUHD to 2'-deoxyinosine substitutions (a minor-groove modification) suggests that POUS binding repositions the POUHD N-terminal "arm".(ABSTRACT TRUNCATED AT 250 WORDS)
Botfield
M C
MC
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Jancso
A
A
Weiss
M A
MA
D000241:Adenosine / Q000031:analogs & derivatives / Q000737:chemistry; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D005801:Genes, Homeobox; D007288:Inosine / Q000031:analogs & derivatives / Q000737:chemistry; D008969:Molecular Sequence Data; D011485:Protein Binding; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
D004268:DNA-Binding Proteins; D014157:Transcription Factors; D007288:Inosine; D001216:Asparagine; D004247:DNA; C010223:N-methyladenosine; C012271:deoxyinosine; D000241:Adenosine