Sensing complex regulatory networks by conformationally controlled hairpin ribozymes

AUTOR(ES)

Najafi-Shoushtari, S. Hani

FONTE

Oxford University Press

RESUMO

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.

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