Sensing complex regulatory networks by conformationally controlled hairpin ribozymes.
The hairpin ribozyme catalyses RNA cleavage by a mechanism utilizing its conformational flexibility during the docking of two independently folded internal loop domains A and B. Based on this mechanism, we designed hairpin ribozyme variants that can be induced or repressed by external effector oligonucleotides influencing the docking process. We incorporated a third domain C to assimilate alternate stable RNA motifs such as a pseudo-half-knot or an internal stem-loop structure. Small sequence changes in domain C allowed targeted switching of ribozyme activity: the same effector oligonucleotide can either serve as an inducer or repressor. The ribozymes were applied to trp leader mRNA, the RNA sequence tightly bound by l-tryptophan-activated trp-RNA-binding attenuation protein (TRAP). When domain C is complementary to this mRNA, ribozyme activity can be altered by annealing trp leader mRNA, then specifically reverted by its TRAP/tryptophan-mediated sequestration. This approach allows to precisely sense the activity status of a protein controlled by its metabolite molecule.
Najafi-Shoushtari
S Hani
SH
Kekule Institut fur Organische Chemie und Biochemie, University of Bonn, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany.
Mayer
Günter
G
Famulok
Michael
M
D000494:Allosteric Regulation; D001426:Bacterial Proteins / Q000378:metabolism; D004789:Enzyme Activation; D005818:Genetic Engineering / Q000379:methods*; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014364:Tryptophan / Q000032:analysis
D001426:Bacterial Proteins; C079092:MtrB protein, Bacteria; D009841:Oligonucleotides; D016337:RNA, Catalytic; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D014157:Transcription Factors; C418481:hairpin ribozyme; D014364:Tryptophan