MAPPING AND MUTAGENESIS OF THE AMINO-TERMINAL TRANSCRIPTIONAL REPRESSION DOMAIN OF THE DROSOPHILA KRUPPEL PROTEIN

We previously demonstrated that the Drosophila Kruppel protein is a tr anscriptional repressor with separable DNA-binding and transcriptional repression activities. In this study, the minimal amino (N)-terminal repression region of the Kruppel protein was defined by transferring r egions of the Kruppel protein to a heterologous DNA-binding protein, t he lacI protein. Fusion of a predicted alpha-helical region from amino acids 62 to 92 in the N terminus of the Kruppel protein was sufficien t to transfer repression activity. This putative alpha-helix has sever al hydrophobic surfaces, as well as a glutamine-rich surface. Mutants containing multiple amino acid substitutions of the glutamine residues demonstrated that this putative or-helical region is essential for re pression activity of a Kruppel protein containing the entire N-termina l and DNA-binding regions. Furthermore, one point mutant with only a s ingle glutamine on this surface altered to lysine abolished the abilit y of the Kruppel protein to repress, indicating the importance of the amino acid at residue 86 for repression. The N terminus also contained an adjacent activation region localized between amino acids 86 and 11 7. Finally, in accordance with predictions from primary amino acid seq uence similarity, a repression region from the Drosophila even-skipped protein, which was six times more potent than that of the Kruppel pro tein in the mammalian cells, was characterized. This segment included a hydrophobic stretch of 11 consecutive alanine residues and a proline -rich region.