Mechanistic basis for RNA aptamer-based induction of TetR.
The TetR aptamer induces TetR controlled gene expression, and represents an interesting tool for application in synthetic biology. We have analysed the mechanistic basis for RNA aptamer-based induction of TetR. The aptamer binds TetR with a high affinity in the order of 10(7)  M(-1), which is similar to operator DNA binding under the used ionic conditions. We identified the binding epitope of the aptamer on TetR, which consists of amino acids T27, N47 and K48 of both monomers, using loss-of-function analysis and electrophoretic mobility shift assays. Tetracycline-induced conformational changes of TetR led to reorientation of the DNA reading head. This movement destroys the composite binding epitope for the aptamer and leads to reduced RNA binding by one order of magnitude. The aptamer can actively displace TetR from the operator DNA; this could be the key factor for its activity in vivo.
Institut für Molekulare Biowissenschaften, Johann Wolfgang Goethe-Universität Frankfurt, Max-von-Laue-Strasse 9, 60438 Frankfurt/Main, Germany.
Markus
M
Steber
Amit
A
Arora
Jan
J
Hofmann
Bernhard
B
Brutschy
Beatrix
B
Suess
D052157:Aptamers, Nucleotide / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D009875:Operator Regions, Genetic; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D052157:Aptamers, Nucleotide; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C083139:Tet O resistance protein, Bacteria; C470187:TetR(B) protein, E coli