Engineering a ligand-dependent RNA transcriptional activator.
RNA has recently been shown to play diverse roles in gene regulation, including the small molecule-dependent inhibition of translation in prokaryotes. To create an artificial genetic switch that acts at the level of transcription, we fused a small molecule binding aptamer to a previously evolved RNA that activates transcription when localized to a promoter. We designed a conformational shift in which a helical element required for transcriptional activation was stabilized upon ligand binding. Selection and screening in S. cerevisiae optimized the linker region, generating an RNA that is 10-fold more active in the presence of tetramethylrosamine (TMR). TMR increases the activity of this evolved RNA in a graded, dose-dependent manner. Our results exemplify a strategy for controlling the activity of laboratory-evolved RNAs in living cells.
Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, USA.
Allen R
AR
Buskirk
Angela
A
Landrigan
David R
DR
Liu
D001483:Base Sequence; D019020:Directed Molecular Evolution; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D004353:Drug Evaluation, Preclinical; D005818:Genetic Engineering; D006575:Heterocyclic Compounds, 3-Ring / Q000737:chemistry / Q000494:pharmacology; D008024:Ligands; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012235:Rhodamines; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation / Q000235:genetics*
D006575:Heterocyclic Compounds, 3-Ring; D008024:Ligands; D012235:Rhodamines; C412715:tetramethylrosamine; D012313:RNA