Limits...
Direct and specific chemical control of eukaryotic translation with a synthetic RNA-protein interaction.

Goldfless SJ, Belmont BJ, de Paz AM, Liu JF, Niles JC - Nucleic Acids Res. (2012)

Bottom Line: Here, we demonstrate the use of a chemically-inducible RNA-protein interaction to regulate eukaryotic translation.By genetically encoding Tet Repressor protein (TetR)-binding RNA elements into the 5'-untranslated region (5'-UTR) of an mRNA, translation of a downstream coding sequence is directly controlled by TetR and tetracycline analogs.In endogenous and synthetic 5'-UTR contexts, this system efficiently regulates the expression of multiple target genes, and is sufficiently stringent to distinguish functional from non-functional RNA-TetR interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.

ABSTRACT
Sequence-specific RNA-protein interactions, though commonly used in biological systems to regulate translation, are challenging to selectively modulate. Here, we demonstrate the use of a chemically-inducible RNA-protein interaction to regulate eukaryotic translation. By genetically encoding Tet Repressor protein (TetR)-binding RNA elements into the 5'-untranslated region (5'-UTR) of an mRNA, translation of a downstream coding sequence is directly controlled by TetR and tetracycline analogs. In endogenous and synthetic 5'-UTR contexts, this system efficiently regulates the expression of multiple target genes, and is sufficiently stringent to distinguish functional from non-functional RNA-TetR interactions. Using a reverse TetR variant, we illustrate the potential for expanding the regulatory properties of the system through protein engineering strategies.

Show MeSH

Related in: MedlinePlus

Placement of TetR-binding RNA aptamers within the 5′-UTR. (a) Illustration of the regulation scheme used to demonstrate functionality of the TetR–aptamer module. TetR binding to the aptamer element within the 5′-UTR of the regulated ORF inhibits its translation. Tetracycline analogs (e.g. aTc and Dox) induce translation by disrupting the TetR–aptamer interaction. (b) The primary and predicted secondary structures of aptamer 5–1.2 (bases 10–60) within the 5′-UTR context used in this study are shown. Residues comprising two conserved regions (Motifs 1 and 2) indispensable to TetR binding are circled. Three potential start codons, 1–3, are shown in green bold italics. Start codon 1 (within Motif 1) is out of frame, whereas 2 is in frame with 3, the downstream ORF’s native start codon.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3351163&req=5

gks028-F1: Placement of TetR-binding RNA aptamers within the 5′-UTR. (a) Illustration of the regulation scheme used to demonstrate functionality of the TetR–aptamer module. TetR binding to the aptamer element within the 5′-UTR of the regulated ORF inhibits its translation. Tetracycline analogs (e.g. aTc and Dox) induce translation by disrupting the TetR–aptamer interaction. (b) The primary and predicted secondary structures of aptamer 5–1.2 (bases 10–60) within the 5′-UTR context used in this study are shown. Residues comprising two conserved regions (Motifs 1 and 2) indispensable to TetR binding are circled. Three potential start codons, 1–3, are shown in green bold italics. Start codon 1 (within Motif 1) is out of frame, whereas 2 is in frame with 3, the downstream ORF’s native start codon.

Mentions: Secondary structure predictions, such as the one shown in Figure 1b, were made using mfold (16). Equilibrium binding constants were determined using a cytometric bead binding assay as described (17).Figure 1.


Direct and specific chemical control of eukaryotic translation with a synthetic RNA-protein interaction.

Goldfless SJ, Belmont BJ, de Paz AM, Liu JF, Niles JC - Nucleic Acids Res. (2012)

Placement of TetR-binding RNA aptamers within the 5′-UTR. (a) Illustration of the regulation scheme used to demonstrate functionality of the TetR–aptamer module. TetR binding to the aptamer element within the 5′-UTR of the regulated ORF inhibits its translation. Tetracycline analogs (e.g. aTc and Dox) induce translation by disrupting the TetR–aptamer interaction. (b) The primary and predicted secondary structures of aptamer 5–1.2 (bases 10–60) within the 5′-UTR context used in this study are shown. Residues comprising two conserved regions (Motifs 1 and 2) indispensable to TetR binding are circled. Three potential start codons, 1–3, are shown in green bold italics. Start codon 1 (within Motif 1) is out of frame, whereas 2 is in frame with 3, the downstream ORF’s native start codon.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351163&req=5

gks028-F1: Placement of TetR-binding RNA aptamers within the 5′-UTR. (a) Illustration of the regulation scheme used to demonstrate functionality of the TetR–aptamer module. TetR binding to the aptamer element within the 5′-UTR of the regulated ORF inhibits its translation. Tetracycline analogs (e.g. aTc and Dox) induce translation by disrupting the TetR–aptamer interaction. (b) The primary and predicted secondary structures of aptamer 5–1.2 (bases 10–60) within the 5′-UTR context used in this study are shown. Residues comprising two conserved regions (Motifs 1 and 2) indispensable to TetR binding are circled. Three potential start codons, 1–3, are shown in green bold italics. Start codon 1 (within Motif 1) is out of frame, whereas 2 is in frame with 3, the downstream ORF’s native start codon.
Mentions: Secondary structure predictions, such as the one shown in Figure 1b, were made using mfold (16). Equilibrium binding constants were determined using a cytometric bead binding assay as described (17).Figure 1.

Bottom Line: Here, we demonstrate the use of a chemically-inducible RNA-protein interaction to regulate eukaryotic translation.By genetically encoding Tet Repressor protein (TetR)-binding RNA elements into the 5'-untranslated region (5'-UTR) of an mRNA, translation of a downstream coding sequence is directly controlled by TetR and tetracycline analogs.In endogenous and synthetic 5'-UTR contexts, this system efficiently regulates the expression of multiple target genes, and is sufficiently stringent to distinguish functional from non-functional RNA-TetR interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.

ABSTRACT
Sequence-specific RNA-protein interactions, though commonly used in biological systems to regulate translation, are challenging to selectively modulate. Here, we demonstrate the use of a chemically-inducible RNA-protein interaction to regulate eukaryotic translation. By genetically encoding Tet Repressor protein (TetR)-binding RNA elements into the 5'-untranslated region (5'-UTR) of an mRNA, translation of a downstream coding sequence is directly controlled by TetR and tetracycline analogs. In endogenous and synthetic 5'-UTR contexts, this system efficiently regulates the expression of multiple target genes, and is sufficiently stringent to distinguish functional from non-functional RNA-TetR interactions. Using a reverse TetR variant, we illustrate the potential for expanding the regulatory properties of the system through protein engineering strategies.

Show MeSH
Related in: MedlinePlus