Competitive regulation of modular allosteric aptazymes by a small molecule and oligonucleotide effector.
The hairpin ribozyme can catalyze the cleavage of RNA substrates by employing its conformational flexibility. To form a catalytic complex, the two domains A and B of the hairpin-ribozyme complex must interact with one another in a folding step called docking. We have constructed hairpin ribozyme variants harboring an aptamer sequence that can be allosterically induced by flavin mononucleotide (FMN). Domains A and B are separated by distinct bridge sequences that communicate the formation of the FMN-aptamer complex to domains A and B, facilitating their docking. In the presence of a short oligonucleotide that is complementary to the aptamer, catalytic activity of the ribozyme is completely abolished, due to the formation of an extended conformer that cannot perform catalysis. However, in the presence of the small molecule effector FMN, the inhibitory effect of the oligonucleotide is competitively neutralized and the ribozyme is activated 150-fold. We thus have established a new principle for the regulation of ribozyme catalysis in which two regulatory factors (an oligonucleotide and a small molecule) that switch the ribozyme's activity in opposite directions compete for the same binding site in the aptamer domain.
Najafi-Shoushtari
S Hani
SH
Kekulé Institut für Organische Chemie und Biochemie, University of Bonn, 53121 Bonn, Germany.
Famulok
Michael
M
D000494:Allosteric Regulation; D000495:Allosteric Site; D020029:Base Pairing; D001483:Base Sequence / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D004789:Enzyme Activation; D005486:Flavin Mononucleotide / Q000378:metabolism*; D005818:Genetic Engineering; D007700:Kinetics; D015394:Molecular Structure; D009154:Mutation; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016337:RNA, Catalytic / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D009841:Oligonucleotides; D016337:RNA, Catalytic; C418481:hairpin ribozyme; D005486:Flavin Mononucleotide