Mapping and mutagenesis of the amino-terminal transcriptional repression domain of the Drosophila Kruppel protein

J. D. Licht, W. Hanna-Rose, J. C. Reddy, M. A. English, M. Ro, M. Grossel, R. Shaknovich, U. Hansen

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Abstract

We previously demonstrated that the Drosophila Kruppel protein is a transcriptional 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 regions of the Kruppel protein to a heterologous DNA-binding protein, the lacI protein. Fusion of a predicted α-helical region from amino acids 62 to 92 in the N terminus of the Kruppel protein was sufficient to transfer repression activity. This putative α-helix has several hydrophobic surfaces, as well as a glutamine-rich surface. Mutants containing multiple amino acid substitutions of the glutamine residues demonstrated that this putative α- helical region is essential for repression activity of a Kruppel protein containing the entire N-terminal and DNA-binding regions. Furthermore, one point mutant with only a single glutamine on this surface altered to lysine abolished the ability 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 117. Finally, in accordance with predictions from primary amino acid sequence similarity, a repression region from the Drosophila even-skipped protein, which was six times more potent than that of the Kruppel protein in the mammalian cells, was characterized. This segment included a hydrophobic stretch of 11 consecutive alanine residues and a proline-rich region.

Original languageEnglish (US)
Pages (from-to)4057-4066
Number of pages10
JournalMolecular and cellular biology
Volume14
Issue number6
DOIs
StatePublished - 1994

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

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