A single glutamic acid residue plays a key role in the transcriptional activation function of lambda repressor.
Previous experiments have suggested that negative charge is an important aspect of the activating region of lambda repressor as it is for at least one class of eukaryotic transcriptional activators. Here we randomize amino acids in the activating region of repressor and assay the function of over 100 variants. We find that acidic residues at the four solvent-exposed positions on the surface of an alpha helix (helix 2 in the structure) together comprise a strong activating region. Only one of these acidic residues, however, is critical for activation, and at this position glutamate is strongly preferred to aspartate. At the three remaining positions, certain uncharged residues (different ones at each position) function as well as or better than the acidic residues. Basic residues, however, are highly detrimental to function at all four positions. Our mutagenesis studies also suggest limitations on amino acid substitutions that allow formation of the helix-turn-helix DNA binding motif found in repressor and in many other DNA binding regulatory proteins.
Bushman
F D
FD
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
Shang
C
C
Ptashne
M
M
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D005796:Genes; D005971:Glutamates; D018698:Glutamic Acid; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009876:Operon; D010957:Plasmids; D011487:Protein Conformation; D012097:Repressor Proteins / Q000235:genetics* / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D005971:Glutamates; D012097:Repressor Proteins; D014157:Transcription Factors; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D018698:Glutamic Acid