Functionally important residues of the Tet repressor inducing peptide TIP determined by a complete mutational analysis.
Tet repressor (TetR) is widely used to control gene expression in pro- and eukaryotes. The mechanism of induction by its natural inducer tetracycline is well characterized. A 16-mer oligopeptide, called TIP, fused to thioredoxin A (TrxA) of Escherichia coli is an artificial inducer of TetR. We analyzed the sequence requirements of TIP by directed and random single amino acid substitutions and identified residues important for TetR induction. An alanine scanning analysis of the first twelve residues showed that all except the ones at position eleven and twelve are important for induction. A randomization of residues at positions one to twelve of TIP revealed the properties of each residue necessary for induction. These further insights into the specificity of TIP-TetR interaction are discussed in the light of the X-ray structure of the [TetR-TIP] complex. The last four residues of TIP contribute indirectly to TetR induction by increasing the steady-state level of the fusion protein. TIP mutants fused N-terminally or C-terminally to TrxA in E. coli induce with the same efficiency indicating identical binding and induction mechanisms, and the lack of contribution from TrxA.
Daam
Janko
J
Lehrstuhl für Mikrobiologie, Department Biologie, Friedrich-Alexander Universität Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Germany.
Mehdaoui
Kaoutar
K
Klotzsche
Marcus
M
Pfleiderer
Klaus
K
Berens
Christian
C
Hillen
Wolfgang
W
D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004252:DNA Mutational Analysis; D029968:Escherichia coli Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D050505:Mutant Proteins / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism
D029968:Escherichia coli Proteins; D050505:Mutant Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C517609:TrxA protein, E coli; C034811:tetracycline resistance-encoding transposon repressor protein; D013879:Thioredoxins; D000409:Alanine