title: Rational design of a synthetic mammalian riboswitch as a ligand-responsive -1 ribosomal frame-shifting stimulator.
abstract: Metabolite-responsive RNA pseudoknots derived from prokaryotic riboswitches have been shown to stimulate -1 programmed ribosomal frameshifting (PRF), suggesting -1 PRF as a promising gene expression platform to extend riboswitch applications in higher eukaryotes. However, its general application has been hampered by difficulty in identifying a specific ligand-responsive pseudoknot that also functions as a ligand-dependent -1 PRF stimulator. We addressed this problem by using the -1 PRF stimulation pseudoknot of SARS-CoV (SARS-PK) to build a ligand-dependent -1 PRF stimulator. In particular, the extra stem of SARS-PK was replaced by an RNA aptamer of theophylline and designed to couple theophylline binding with the stimulation of -1 PRF. Conformational and functional analyses indicate that the engineered theophylline-responsive RNA functions as a mammalian riboswitch with robust theophylline-dependent -1 PRF stimulation activity in a stable human 293T cell-line. Thus, RNA-ligand interaction repertoire provided by in vitro selection becomes accessible to ligand-specific -1 PRF stimulator engineering using SARS-PK as the scaffold for synthetic biology application.
authors: Institute of Biochemistry, National Chung-Hsing University, 250 Kuo-Kung Road, Taichung, 402 Taiwan.
authors: Ya-Hui
authors: YH
authors: Lin
authors: Institute of Biochemistry, National Chung-Hsing University, 250 Kuo-Kung Road, Taichung, 402 Taiwan kychang@dragon.nchu.edu.tw.
authors: Kung-Yao
authors: KY
authors: Chang
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D018965:Frameshifting, Ribosomal / Q000187:drug effects; D006801:Humans; D055029:Inverted Repeat Sequences; D008024:Ligands; D008322:Mammals; D008954:Models, Biological; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics; D058928:Riboswitch / Q000187:drug effects; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics; D058615:Synthetic Biology; D013806:Theophylline / Q000737:chemistry / Q000494:pharmacology
chemical_list: D008024:Ligands; D012367:RNA, Viral; D058928:Riboswitch; D013806:Theophylline