Rational design of a synthetic mammalian riboswitch as a ligand-responsive -1 ribosomal frame-shifting stimulator

Lin, Ya-Hui; Chang, Kung-Yao

Recently, metabolite-binding units of some prokaryotic riboswitches grafted into an open reading frame (ORF) have been shown to stimulate −1 programmed ribosomal frameshifting (PRF) in response to specific metabolites, suggesting that −1 PRF holds promise as an expression platform for the implementation of an engineered mammalian riboswitch (8, 9) . The −1 PRF involves the backward movement of an elongating ribosome by one nucleotide relative to the decoding reading-frame. It leads to a switch of the decoding process into a −1 reading-frame to generate a protein with its C-terminal domain composition being determined by the new reading-frame. It has been adopted in a variety of viruses to control the ratio between viral proteins crucial for optimal propagation via instrumental frameshifting efficiency (10, 11) . −1 PRF occurs on a shifty sequence with a low basal efficiency and can be further enhanced by an RNA structure optimally positioned downstream of the shifty sequence (12) . The downstream RNA structure is usually an H-type pseudoknot (13) composed of an RNA hairpin with its loop sequences pairing with complementary sequences downstream of the hairpin stem (stem 1) to form a second duplex (stem 2). Given the critical role of a downstream stimulator in the efficiency of eukaryotic −1 PRF, the ability to modulate stimulator conformation formation by a ligand-binding RNA aptamer could result in a ligand-responsive −1 PRF stimulator. However, only a subset of the H-type pseudoknot can stimulate −1 PRF efficiently.