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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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Positive/negative selection of yeast mediated by TetR aptamers. (a) Illustration of the URA3 positive/negative selection scheme showing the predicted growth phenotypes of the strains under indicated conditions. (b) TetR-expressing strains in which Ura3p synthesis is controlled by either 5–1.2 or 5–1.2m2 grow similarly on YPD. (c) Negative selection on 5-FOA only permits growth of strains capable of repressing Ura3p translation. (d, e) When 5–1.2 controls Ura3p translation, aTc is required for growth in the absence of uracil. In each condition, 10-fold dilutions are shown from left to right. Plates were imaged after growth at 30°C for 2 days (YPD) or 3 days (synthetic defined media).
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gks028-F4: Positive/negative selection of yeast mediated by TetR aptamers. (a) Illustration of the URA3 positive/negative selection scheme showing the predicted growth phenotypes of the strains under indicated conditions. (b) TetR-expressing strains in which Ura3p synthesis is controlled by either 5–1.2 or 5–1.2m2 grow similarly on YPD. (c) Negative selection on 5-FOA only permits growth of strains capable of repressing Ura3p translation. (d, e) When 5–1.2 controls Ura3p translation, aTc is required for growth in the absence of uracil. In each condition, 10-fold dilutions are shown from left to right. Plates were imaged after growth at 30°C for 2 days (YPD) or 3 days (synthetic defined media).

Mentions: Different implementations of the translation regulatory system may necessitate discovering TetR aptamers with unique binding characteristics. Therefore, it is important to define a strategy for rapidly identifying new functional aptamer variants. To support this effort, we devised a positive/negative selection scheme capable of distinguishing between TetR-binding and non-binding RNA sequences (Figure 4a). To establish proof-of-concept, we constructed plasmids containing the yeast URA3 gene (encoding orotidine-5′-phosphate decarboxylase, Ura3p) with either 5–1.2 or 5–1.2m2 within the 5′-UTR context used earlier. We co-transformed each separately with a TetR-encoding plasmid into a yeast ura3 mutant auxotrophic for uracil. These strains exhibited similar growth on nutrient-rich YPD media (Figure 4b). For the negative selection, we plated cells on media containing 5-fluoroorotic acid (5-FOA), which is converted to the cytotoxic 5-fluorouracil by Ura3p. These conditions only permit growth of cells containing a functional TetR/5–1.2 interaction that can repress Ura3p synthesis (Figure 4c). For the positive selection, we plated cells in the presence of aTc on uracil-deficient media. Cells surviving this selection step have been induced to synthesize Ura3p to complement the uracil auxotrophy. This ensures that interactions identified by negative selection are aTc inducible (Figure 4d). As expected, the cells containing a functional TetR–aptamer system exhibit a significant growth defect when plated in the absence of aTc on uracil-deficient media (Figure 4e). To further establish the utility of this selection scheme, we mixed TetR-expressing cells containing URA3 controlled by either 5–1.2 or 5–1.2m2 in a ratio of 1:104, respectively. From this mixture, we plated ∼1.5 × 105 cells on media containing 5-FOA, and 17 large colonies were grown. Sequencing the 5′-UTR of plasmid DNA isolated from 10 of these colonies revealed that all carried the 5–1.2 sequence. These data demonstrate that this selection strategy can specifically recover functional TetR aptamers from a large non-functional background, which should prove useful for identifying aptamer or TetR variants with novel regulatory characteristics.Figure 4.


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)

Positive/negative selection of yeast mediated by TetR aptamers. (a) Illustration of the URA3 positive/negative selection scheme showing the predicted growth phenotypes of the strains under indicated conditions. (b) TetR-expressing strains in which Ura3p synthesis is controlled by either 5–1.2 or 5–1.2m2 grow similarly on YPD. (c) Negative selection on 5-FOA only permits growth of strains capable of repressing Ura3p translation. (d, e) When 5–1.2 controls Ura3p translation, aTc is required for growth in the absence of uracil. In each condition, 10-fold dilutions are shown from left to right. Plates were imaged after growth at 30°C for 2 days (YPD) or 3 days (synthetic defined media).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks028-F4: Positive/negative selection of yeast mediated by TetR aptamers. (a) Illustration of the URA3 positive/negative selection scheme showing the predicted growth phenotypes of the strains under indicated conditions. (b) TetR-expressing strains in which Ura3p synthesis is controlled by either 5–1.2 or 5–1.2m2 grow similarly on YPD. (c) Negative selection on 5-FOA only permits growth of strains capable of repressing Ura3p translation. (d, e) When 5–1.2 controls Ura3p translation, aTc is required for growth in the absence of uracil. In each condition, 10-fold dilutions are shown from left to right. Plates were imaged after growth at 30°C for 2 days (YPD) or 3 days (synthetic defined media).
Mentions: Different implementations of the translation regulatory system may necessitate discovering TetR aptamers with unique binding characteristics. Therefore, it is important to define a strategy for rapidly identifying new functional aptamer variants. To support this effort, we devised a positive/negative selection scheme capable of distinguishing between TetR-binding and non-binding RNA sequences (Figure 4a). To establish proof-of-concept, we constructed plasmids containing the yeast URA3 gene (encoding orotidine-5′-phosphate decarboxylase, Ura3p) with either 5–1.2 or 5–1.2m2 within the 5′-UTR context used earlier. We co-transformed each separately with a TetR-encoding plasmid into a yeast ura3 mutant auxotrophic for uracil. These strains exhibited similar growth on nutrient-rich YPD media (Figure 4b). For the negative selection, we plated cells on media containing 5-fluoroorotic acid (5-FOA), which is converted to the cytotoxic 5-fluorouracil by Ura3p. These conditions only permit growth of cells containing a functional TetR/5–1.2 interaction that can repress Ura3p synthesis (Figure 4c). For the positive selection, we plated cells in the presence of aTc on uracil-deficient media. Cells surviving this selection step have been induced to synthesize Ura3p to complement the uracil auxotrophy. This ensures that interactions identified by negative selection are aTc inducible (Figure 4d). As expected, the cells containing a functional TetR–aptamer system exhibit a significant growth defect when plated in the absence of aTc on uracil-deficient media (Figure 4e). To further establish the utility of this selection scheme, we mixed TetR-expressing cells containing URA3 controlled by either 5–1.2 or 5–1.2m2 in a ratio of 1:104, respectively. From this mixture, we plated ∼1.5 × 105 cells on media containing 5-FOA, and 17 large colonies were grown. Sequencing the 5′-UTR of plasmid DNA isolated from 10 of these colonies revealed that all carried the 5–1.2 sequence. These data demonstrate that this selection strategy can specifically recover functional TetR aptamers from a large non-functional background, which should prove useful for identifying aptamer or TetR variants with novel regulatory characteristics.Figure 4.

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