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Biological Procedures Online (1)
Journal of molecular biology (1)
Liu, Ying (2)
Bondos, Sarah E (1)
Bondos, Sarah E. (1)
Gonzalez, Kim L (1)
Hsiao, Hao-Ching (1)
Matthews, Kathleen S (1)
Matthews, Kathleen S. (1)
Merchant, Zabeena (1)
Year of Publication
INTERNAL REGULATORY INTERACTIONS DETERMINE DNA BINDING SPECIFICITY BY A HOX TRANSCRIPTION FACTOR
Matthews, Kathleen S.
Journal of molecular biology
In developing bilaterans, the Hox transcription factor family regulates batteries of downstream genes to diversify serially repeated units. Given Hox homeodomains bind a wider array of DNA binding sites in vitro than are regulated by the full-length protein in vivo, regions outside the homeodomain must aid DNA site selection. Indeed, we find affinity for disparate DNA sequences varies less than 3-fold for the homeodomain isolated from the Drosophila Hox protein Ultrabithorax Ia (UbxHD), whereas for the full-length protein (UbxIa) affinity differs by more than 10-fold. The rank order of preferred DNA sequences also differs, further demonstrating distinct DNA binding preferences. The increased specificity of UbxIa can be partially attributed to the I1 region, which lies adjacent to the homeodomain and directly impacts binding energetics. Each of three segments within I1 – the Extradenticle-binding YPWM motif, the 6 amino acids immediately N-terminal to this motif, and the 8 amino acids abutting the YPWM C-terminus – uniquely contribute to DNA specificity. Combination of these regions synergistically modifies DNA binding to further enhance specificity. Intriguingly, the presence of the YPWM motif in UbxIa inhibits DNA binding only to Ubx•Extradenticle heterodimer binding sites, potentially functioning in vivo to prevent Ubx monomers from binding and misregulating heterodimer target genes. However, removal of the surrounding region allows the YPWM motif to also inhibit binding to Hox-only recognition sequences. Despite a modular domain design for Hox proteins, these results suggest that multiple Hox protein regions form a network of regulatory interactions that coordinate context-and gene-specific responses. Since most non-homeodomain regions are not conserved between Hox family members, these regulatory interactions have the potential to diversify binding by the highly homologous Hox homeodomains.
Hox; DNA binding; homeodomain; YPWM; hexapeptide motif
Media composition influences yeast one- and two-hybrid results
Gonzalez, Kim L
Matthews, Kathleen S
Biological Procedures Online
Although yeast two-hybrid experiments are commonly used to identify protein interactions, the frequent occurrence of false negatives and false positives hampers data interpretation. Using both yeast one-hybrid and two-hybrid experiments, we have identified potential sources of these problems: the media preparation protocol and the source of the yeast nitrogen base may not only impact signal range but also effect whether a result appears positive or negative. While altering media preparation may optimize signal differences for individual experiments, media preparation must be reported in detail to replicate studies and accurately compare results from different experiments.
Yeast one-hybrid; yeast two-hybrid; protein-protein interaction; accuracy; false positive; false negative
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