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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.

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Inversion of Dox-inducible phenotype with a mutant TetR. (a) A revTetR variant dose-dependently represses RRL translation of synthetic FLuc mRNA as in Figure 2c, but only in the presence of Dox. (b) A revTetR variant specifically represses translation as in Figure 2d, but only in the presence of Dox. In all figures, data represent the mean ± SD of at least four experiments. A two-tailed, unpaired t-test was used to calculate the significance (α = 0.005) of the difference between induced and uninduced conditions. ¥P = 8.1 × 10−5.
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gks028-F5: Inversion of Dox-inducible phenotype with a mutant TetR. (a) A revTetR variant dose-dependently represses RRL translation of synthetic FLuc mRNA as in Figure 2c, but only in the presence of Dox. (b) A revTetR variant specifically represses translation as in Figure 2d, but only in the presence of Dox. In all figures, data represent the mean ± SD of at least four experiments. A two-tailed, unpaired t-test was used to calculate the significance (α = 0.005) of the difference between induced and uninduced conditions. ¥P = 8.1 × 10−5.

Mentions: For cell-free translation with rabbit reticulocyte lysate (RRL), the mRNA concentration was adjusted to 133 nM in RBBD (RBB plus 1 mM dithiothreitol and 10 μg/ml bovine serum albumin, New England Biolabs) and refolded by heating at 65°C for 2 min and incubating at room temperature for 10 min. TetR(B) with N-terminal T7 and C-terminal His6 tags, and revTetR-S2 with a C-terminal His6 tag were purified from Escherichia coli as previously described (17). TetR and revTetR-S2 dilutions were prepared in RBBD with or without 22 µM doxycycline (Dox). For each translation reaction, 1.5 μl mRNA was mixed with 2 μl TetR or revTetR-S2, allowed to equilibrate for 30 min at room temperature, and then mixed with 0.5 µl of a complete amino acid solution (0.5 mM each amino acid, Promega) and 6 μl of nuclease-treated RRL (Promega). The final reactions contained 20 nM mRNA, 0–300 nM repressor protein and 25 µM amino acids in 0.6 × RRL. Reactions were incubated at 30°C for 20 min. For the screening experiment in Figure 2b, reactions were stopped by adding 200 μl Stop Buffer [20 mM GlyGly-NaOH, pH 7.8, 8 mM magnesium acetate, 0.13 mM EDTA, 500 µM cycloheximide]. FLuc activity was determined by mixing 90 μl of the reaction mixture with 40 μl FLuc Assay Buffer [20 mM GlyGly–NaOH, pH 7.8, 90 mM dithiothreitol (DTT), 8 mM Mg(OAc)2, 0.13 mM EDTA, 1.5 mM ATP, 0.8 mM coenzyme A, 1.4 mM D-luciferin] in a 96-well microplate and measuring luminescence with a Spectramax L plate reader (Molecular Devices). Translation activity was calculated as the intensity of FLuc signal. For the TetR and RevTetR(S2) titration experiments in Figures 2c and 5a, a Renilla luciferase mRNA lacking an aptamer was included in each translation reaction as a reference. Reactions were performed as described above, and stopped with the addition of 200 μl of 500 μM cycloheximide in water. Four microliters (4µ L) of the reaction mixture was mixed with 20 μl of passive lysis buffer (Promega) and dual luciferase activity was measured by the sequential addition of 100 μl DLB1 (75 mM HEPES-K, 20 mM DTT, 4 mM MgSO4, 0.1 mM EDTA, 0.53 mM ATP, 0.27 mM coenzyme A, 0.47 mM D-luciferin, pH 8.0) and 100 μl DLB2 [15 mM Na4P2O7, 7.5 mM sodium acetate, 10 mM EDTA, 400 mM Na2SO4, 1% (v/v) methanol, 10 μM 2-(4′-(dimethylamino)phenyl)-6-methyl-benzothiazole, 5 mM KI, 12 μM benzyl coelenterazine, pH 5.0]. Translation was calculated as the ratio of FLuc (aptamer-regulated) to RLuc (aptamer-independent) signal.Figure 2.


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)

Inversion of Dox-inducible phenotype with a mutant TetR. (a) A revTetR variant dose-dependently represses RRL translation of synthetic FLuc mRNA as in Figure 2c, but only in the presence of Dox. (b) A revTetR variant specifically represses translation as in Figure 2d, but only in the presence of Dox. In all figures, data represent the mean ± SD of at least four experiments. A two-tailed, unpaired t-test was used to calculate the significance (α = 0.005) of the difference between induced and uninduced conditions. ¥P = 8.1 × 10−5.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks028-F5: Inversion of Dox-inducible phenotype with a mutant TetR. (a) A revTetR variant dose-dependently represses RRL translation of synthetic FLuc mRNA as in Figure 2c, but only in the presence of Dox. (b) A revTetR variant specifically represses translation as in Figure 2d, but only in the presence of Dox. In all figures, data represent the mean ± SD of at least four experiments. A two-tailed, unpaired t-test was used to calculate the significance (α = 0.005) of the difference between induced and uninduced conditions. ¥P = 8.1 × 10−5.
Mentions: For cell-free translation with rabbit reticulocyte lysate (RRL), the mRNA concentration was adjusted to 133 nM in RBBD (RBB plus 1 mM dithiothreitol and 10 μg/ml bovine serum albumin, New England Biolabs) and refolded by heating at 65°C for 2 min and incubating at room temperature for 10 min. TetR(B) with N-terminal T7 and C-terminal His6 tags, and revTetR-S2 with a C-terminal His6 tag were purified from Escherichia coli as previously described (17). TetR and revTetR-S2 dilutions were prepared in RBBD with or without 22 µM doxycycline (Dox). For each translation reaction, 1.5 μl mRNA was mixed with 2 μl TetR or revTetR-S2, allowed to equilibrate for 30 min at room temperature, and then mixed with 0.5 µl of a complete amino acid solution (0.5 mM each amino acid, Promega) and 6 μl of nuclease-treated RRL (Promega). The final reactions contained 20 nM mRNA, 0–300 nM repressor protein and 25 µM amino acids in 0.6 × RRL. Reactions were incubated at 30°C for 20 min. For the screening experiment in Figure 2b, reactions were stopped by adding 200 μl Stop Buffer [20 mM GlyGly-NaOH, pH 7.8, 8 mM magnesium acetate, 0.13 mM EDTA, 500 µM cycloheximide]. FLuc activity was determined by mixing 90 μl of the reaction mixture with 40 μl FLuc Assay Buffer [20 mM GlyGly–NaOH, pH 7.8, 90 mM dithiothreitol (DTT), 8 mM Mg(OAc)2, 0.13 mM EDTA, 1.5 mM ATP, 0.8 mM coenzyme A, 1.4 mM D-luciferin] in a 96-well microplate and measuring luminescence with a Spectramax L plate reader (Molecular Devices). Translation activity was calculated as the intensity of FLuc signal. For the TetR and RevTetR(S2) titration experiments in Figures 2c and 5a, a Renilla luciferase mRNA lacking an aptamer was included in each translation reaction as a reference. Reactions were performed as described above, and stopped with the addition of 200 μl of 500 μM cycloheximide in water. Four microliters (4µ L) of the reaction mixture was mixed with 20 μl of passive lysis buffer (Promega) and dual luciferase activity was measured by the sequential addition of 100 μl DLB1 (75 mM HEPES-K, 20 mM DTT, 4 mM MgSO4, 0.1 mM EDTA, 0.53 mM ATP, 0.27 mM coenzyme A, 0.47 mM D-luciferin, pH 8.0) and 100 μl DLB2 [15 mM Na4P2O7, 7.5 mM sodium acetate, 10 mM EDTA, 400 mM Na2SO4, 1% (v/v) methanol, 10 μM 2-(4′-(dimethylamino)phenyl)-6-methyl-benzothiazole, 5 mM KI, 12 μM benzyl coelenterazine, pH 5.0]. Translation was calculated as the ratio of FLuc (aptamer-regulated) to RLuc (aptamer-independent) signal.Figure 2.

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