Dataset Information

Inducible gene expression from the plastid genome by a synthetic riboswitch.

ABSTRACT: Riboswitches are natural RNA sensors that regulate gene expression in response to ligand binding. Riboswitches have been identified in prokaryotes and eukaryotes but are unknown in organelles (mitochondria and plastids). Here we have tested the possibility to engineer riboswitches for plastids (chloroplasts), a genetic system that largely relies on translational control of gene expression. To this end, we have used bacterial riboswitches and modified them in silico to meet the requirements of translational regulation in plastids. These engineered switches were then tested for functionality in vivo by stable transformation of the tobacco chloroplast genome. We report the identification of a synthetic riboswitch that functions as an efficient translational regulator of gene expression in plastids in response to its exogenously applied ligand theophylline. This riboswitch provides a novel tool for plastid genome engineering that facilitates the tightly regulated inducible expression of chloroplast genes and transgenes and thus has wide applications in functional genomics and biotechnology.

SUBMITTER: Verhounig A

PROVIDER: S-EPMC2852001 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Inducible gene expression from the plastid genome by a synthetic riboswitch.

Verhounig Andreas A Karcher Daniel D Bock Ralph R

Proceedings of the National Academy of Sciences of the United States of America 20100322 14

Riboswitches are natural RNA sensors that regulate gene expression in response to ligand binding. Riboswitches have been identified in prokaryotes and eukaryotes but are unknown in organelles (mitochondria and plastids). Here we have tested the possibility to engineer riboswitches for plastids (chloroplasts), a genetic system that largely relies on translational control of gene expression. To this end, we have used bacterial riboswitches and modified them in silico to meet the requirements of tr ...[more]

PMID: 20308585

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Project description:Robust genetic systems for the hyperthermophilic Thermococcales have facilitated the overexpression of native genes, enabled the addition of sequences encoding secretion signals, epitope, and affinity tags to coding regions, and aided the introduction of sequences encoding new proteins in these fast-growing fermentative heterotrophs. However, tightly controlled and easily manipulated systems facilitating regulated gene expression are limited for these hosts. Here, we describe an alternative method for regulatory control reliant on a cis-encoded functional riboswitch in the model archaeon Thermococcus kodakarensis Despite the hyperthermophilic growth temperatures, the proposed structure of the riboswitch conforms to a fluoride-responsive riboswitch encoded in many bacteria and similarly functions to regulate a component-conserved fluoride export pathway. Deleting components of the fluoride export pathway generates T. kodakarensis strains with increased fluoride sensitivity. The mechanism underlying regulated expression suggested that the riboswitch-encoding sequences could be utilized as a tunable expression cassette. When appended to a reporter gene, the riboswitch-mediated control system provides fluoride-dependent tunable regulatory potential, offering an alternative system for regulating gene expression. Riboswitch-regulated expression is thus ubiquitous in extant life and can be exploited to generate regulated expression systems for hyperthermophiles.IMPORTANCE Gene expression is controlled by a myriad of interconnected mechanisms that interpret metabolic states and environmental cues to balance cell physiology. Transcription regulation in Archaea is known to employ both typical repressors-operators and transcription activators to regulate transcription initiation in addition to the regulation afforded by chromatin structure. It was perhaps surprising that the presumed ancient mechanism of riboswitch-mediated regulation is found in Bacteria and Eukarya, but seemingly absent in Archaea We demonstrate here that a fluoride-responsive riboswitch functions to regulate a detoxification pathway in the hyperthermophilic archaeon Thermococcus kodakarensis The results obtained define a universal role for riboswitch-mediated regulation, adumbrate the presence of several riboswitch-regulated genes in Thermococcus kodakarensis, demonstrate the utility of RNA-based regulation at high temperatures, and provide a novel riboswitch-regulated expression system to employ in hyperthermophiles.

Dataset Information

Inducible gene expression from the plastid genome by a synthetic riboswitch.

Publications

Inducible gene expression from the plastid genome by a synthetic riboswitch.

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