Mapping and mutagenesis of the amino-terminal transcriptional repression domain of the Drosophila Krüppel protein.
We previously demonstrated that the Drosophila Krüppel 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 Krüppel protein was defined by transferring regions of the Krüppel protein to a heterologous DNA-binding protein, the lacI protein. Fusion of a predicted alpha-helical region from amino acids 62 to 92 in the N terminus of the Krüppel protein was sufficient to transfer repression activity. This putative alpha-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 alpha-helical region is essential for repression activity of a Krüppel 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 Krüppel 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 Krüppel protein in the mammalian cells, was characterized. This segment included a hydrophobic stretch of 11 consecutive alanine residues and a proline-rich region.
Licht
J D
JD
Laboratory of Eukaryotic Transcription, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115.
Hanna-Rose
W
W
Reddy
J C
JC
English
M A
MA
Ro
M
M
Grossel
M
M
Shaknovich
R
R
Hansen
U
U
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D004330:Drosophila / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins; D051741:Kruppel-Like Transcription Factors; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012097:Repressor Proteins; D014157:Transcription Factors / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*
D017931:DNA Primers; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; C497244:Kr protein, Drosophila; D051741:Kruppel-Like Transcription Factors; D012097:Repressor Proteins; D014157:Transcription Factors