title: Tunable Riboregulator Switches for Post-transcriptional Control of Gene Expression.
abstract: Until recently, engineering strategies for altering gene expression have focused on transcription control using strong inducible promoters or one of several methods to knock down wasteful genes. Recently, synthetic riboregulators have been developed for translational regulation of gene expression. Here, we report a new modular synthetic riboregulator class that has the potential to finely tune protein expression and independently control the concentration of each enzyme in an engineered metabolic pathway. This development is important because the most straightforward approach to altering the flux through a particular metabolic step is to increase or decrease the concentration of the enzyme. Our design includes a cis-repressor at the 5' end of the mRNA that forms a stem-loop helix, occluding the ribosomal binding sequence and blocking translation. A trans-expressed activating-RNA frees the ribosomal-binding sequence, which turns on translation. The overall architecture of the riboregulators is designed using Watson-Crick base-pairing stability. We describe here a cis-repressor that can completely shut off translation of antibiotic-resistance reporters and a trans-activator that restores translation. We have established that it is possible to use these riboregulators to achieve translational control of gene expression over a wide dynamic range. We have also found that a targeting sequence can be modified to develop riboregulators that can, in principle, independently regulate translation of many genes. In a selection experiment, we demonstrated that by subtly altering the sequence of the trans-activator it is possible to alter the ratio of the repressed and activated states and to achieve intermediate translational control.
authors.affiliation: Bioenergy and Biome Sciences, Bioscience Division, ‡Theoretical Biology and Biophysics, Theoretical Division, Los Alamos National Laboratory , Los Alamos, New Mexico 87545, United States.
authors.forename: Malathy
authors.initials: M
authors.lastname: Krishnamurthy
authors.affiliation: Bioenergy and Biome Sciences, Bioscience Division, ‡Theoretical Biology and Biophysics, Theoretical Division, Los Alamos National Laboratory , Los Alamos, New Mexico 87545, United States.
authors.forename: Scott P
authors.initials: SP
authors.lastname: Hennelly
authors.affiliation: Bioenergy and Biome Sciences, Bioscience Division, ‡Theoretical Biology and Biophysics, Theoretical Division, Los Alamos National Laboratory , Los Alamos, New Mexico 87545, United States.
authors.forename: Taraka
authors.initials: T
authors.lastname: Dale
authors.affiliation: Bioenergy and Biome Sciences, Bioscience Division, ‡Theoretical Biology and Biophysics, Theoretical Division, Los Alamos National Laboratory , Los Alamos, New Mexico 87545, United States.
authors.forename: Shawn R
authors.initials: SR
authors.lastname: Starkenburg
authors.affiliation: Bioenergy and Biome Sciences, Bioscience Division, ‡Theoretical Biology and Biophysics, Theoretical Division, Los Alamos National Laboratory , Los Alamos, New Mexico 87545, United States.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Martí-Arbona
authors.affiliation: Bioenergy and Biome Sciences, Bioscience Division, ‡Theoretical Biology and Biophysics, Theoretical Division, Los Alamos National Laboratory , Los Alamos, New Mexico 87545, United States.
authors.forename: David T
authors.initials: DT
authors.lastname: Fox
authors.affiliation: Bioenergy and Biome Sciences, Bioscience Division, ‡Theoretical Biology and Biophysics, Theoretical Division, Los Alamos National Laboratory , Los Alamos, New Mexico 87545, United States.
authors.forename: Scott N
authors.initials: SN
authors.lastname: Twary
authors.affiliation: Bioenergy and Biome Sciences, Bioscience Division, ‡Theoretical Biology and Biophysics, Theoretical Division, Los Alamos National Laboratory , Los Alamos, New Mexico 87545, United States.
authors.forename: Karissa Y
authors.initials: KY
authors.lastname: Sanbonmatsu
authors.affiliation: Bioenergy and Biome Sciences, Bioscience Division, ‡Theoretical Biology and Biophysics, Theoretical Division, Los Alamos National Laboratory , Los Alamos, New Mexico 87545, United States.
authors.forename: Clifford J
authors.initials: CJ
authors.lastname: Unkefer
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis / Q000235:genetics*; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D058928:Riboswitch / Q000235:genetics*
chemical_list: D012329:RNA, Bacterial; D058928:Riboswitch
keywords: gene expression; pathway engineering; riboregulators; synthetic biology; translational control