Functional roles of amino acid residues involved in forming the alpha-helix-turn-alpha-helix operator DNA binding motif of Tet repressor from Tn10.
The Tn10 derived Tet repressor contains an amino acid segment with high homology to the alpha-helix-turn-alpha-helix motif (HTH) of other DNA binding proteins. The five most conserved amino acids in HTH are probably involved in structural formation of the motif. Their functional role was probed by saturation mutagenesis yielding 95 single amino acid replacement mutants of Tet repressor. Their binding efficiencies to tet operator were quantitatively determined in vivo. All functional mutants contain amino acid substitutions consistent with their proposed role in a HTH. In particular, only the two smallest amino acids (serine, glycine) can substitute a conserved alanine in the proposed first alpha-helix without loss of activity. The last position of the first alpha-helix, the second position in the turn, and the fourth position in the second alpha-helix require mostly hydrophobic residues. The proposed C-terminus of the first alpha-helix is supported by a more active asparagine compared to glutamine replacement mutant of the wt leucine residue. The turn is located close to the protein surface as indicated by functional lysine and arginine replacements for valine. A glycine residue at the first position in the turn can be replaced by any amino acid yielding mutants with at least residual tet operator affinity. A structural model of the HTH of Tet repressor is presented.
Baumeister
R
R
Lehrstuhl für Mikrobiologie, Friedrich-Alexander Universität Erlangen-Nürnberg, Federal Republic of Germany.
Müller
G
G
Hecht
B
B
Hillen
W
W
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000502:physiology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004274:DNA, Recombinant / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005998:Glycine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009875:Operator Regions, Genetic; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000596:Amino Acids; D001426:Bacterial Proteins; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D005998:Glycine