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A modular strategy for engineering orthogonal chimeric RNA transcription regulators.

Takahashi MK, Lucks JB - Nucleic Acids Res. (2013)

Bottom Line: A comprehensive orthogonality test of these culminated in a 7 × 7 matrix of mutually orthogonal regulators.A comparison between all chimeras tested led to design principles that will facilitate further engineering of orthogonal RNA transcription regulators, and may help elucidate general principles of non-coding RNA regulation.We anticipate that our strategy will accelerate the development of even larger families of orthogonal RNA transcription regulators, and thus create breakthroughs in our ability to construct increasingly sophisticated RNA genetic circuitry.

View Article: PubMed Central - PubMed

Affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA.

ABSTRACT
Antisense RNA transcription attenuators are a key component of the synthetic biology toolbox, with their ability to serve as building blocks for both signal integration logic circuits and transcriptional cascades. However, a central challenge to building more sophisticated RNA genetic circuitry is creating larger families of orthogonal attenuators that function independently of each other. Here, we overcome this challenge by developing a modular strategy to create chimeric fusions between the engineered transcriptional attenuator from plasmid pT181 and natural antisense RNA translational regulators. Using in vivo gene expression assays in Escherichia coli, we demonstrate our ability to create chimeric attenuators by fusing sequences from five different translational regulators. Mutagenesis of these functional attenuators allowed us to create a total of 11 new chimeric attenutaors. A comprehensive orthogonality test of these culminated in a 7 × 7 matrix of mutually orthogonal regulators. A comparison between all chimeras tested led to design principles that will facilitate further engineering of orthogonal RNA transcription regulators, and may help elucidate general principles of non-coding RNA regulation. We anticipate that our strategy will accelerate the development of even larger families of orthogonal RNA transcription regulators, and thus create breakthroughs in our ability to construct increasingly sophisticated RNA genetic circuitry.

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Antisense-RNA transcription and translation control. (A) A transcriptional attenuator that lies in the 5′ untranslated region of a transcript can fold into a structure that allows transcription (ON) when antisense RNA is not present. Antisense RNA binding to the transcribed attenuator region results in the formation of a terminator hairpin, stopping transcription before the gene of interest (OFF, indicated by x symbol) (13). (B, C) Antisense RNA translational control works similarly, though antisense binding results in structures that occlude the ribosome-binding site (RBS) upstream of the gene-coding sequence to block translation (depicted by x symbols). Antisense binding can occur in a loop–loop mechanism where both antisense RNA and target mRNA are in the form of hairpin structures upon interaction (B), or a loop–linear mechanism, where either the antisense RNA or the target mRNA is unstructured (or linear) (C). (D, E) Chimeric transcription attenuators are engineered in this work by replacing portions of the S. aureus pT181 transcriptional attenuator with RNA-binding regions from natural translational regulators. (A–E) Break symbols indicate additional RNA sequence and structure not shown in the cartoons.
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gkt452-F1: Antisense-RNA transcription and translation control. (A) A transcriptional attenuator that lies in the 5′ untranslated region of a transcript can fold into a structure that allows transcription (ON) when antisense RNA is not present. Antisense RNA binding to the transcribed attenuator region results in the formation of a terminator hairpin, stopping transcription before the gene of interest (OFF, indicated by x symbol) (13). (B, C) Antisense RNA translational control works similarly, though antisense binding results in structures that occlude the ribosome-binding site (RBS) upstream of the gene-coding sequence to block translation (depicted by x symbols). Antisense binding can occur in a loop–loop mechanism where both antisense RNA and target mRNA are in the form of hairpin structures upon interaction (B), or a loop–linear mechanism, where either the antisense RNA or the target mRNA is unstructured (or linear) (C). (D, E) Chimeric transcription attenuators are engineered in this work by replacing portions of the S. aureus pT181 transcriptional attenuator with RNA-binding regions from natural translational regulators. (A–E) Break symbols indicate additional RNA sequence and structure not shown in the cartoons.

Mentions: However, the ability of ncRNAs to act as fundamental building blocks of genetic circuits is only beginning to be explored. In prokaryotes, transcription attenuation offers a particularly attractive mechanism for creating RNA genetic circuitry. Transcription attenuators are ncRNAs that control the fate of transcription elongation in response to an input antisense RNA (12,13) (Figure 1A). The attenuator lies in the 5′-untranslated region of a transcript and is thought to fold into two different RNA structures during transcription that either allow (ON) or block (OFF) further elongation by RNA polymerase (12,13). An interaction with a complementary antisense RNA biases the fold to the OFF state, enabling the attenuator to act as a transcriptional switch that senses and responds to antisense RNA signals (Figure 1A). Leveraging this ability to use an RNA input to regulate an RNA output, attenuators built from the Staphylococcus aureus plasmid pT181 were recently configured in simple architectures that evaluated genetic not-or (NOR) logics, and in transcriptional cascades that propagated signals directly as antisense RNA molecules with no intermediate protein species (10).Figure 1.


A modular strategy for engineering orthogonal chimeric RNA transcription regulators.

Takahashi MK, Lucks JB - Nucleic Acids Res. (2013)

Antisense-RNA transcription and translation control. (A) A transcriptional attenuator that lies in the 5′ untranslated region of a transcript can fold into a structure that allows transcription (ON) when antisense RNA is not present. Antisense RNA binding to the transcribed attenuator region results in the formation of a terminator hairpin, stopping transcription before the gene of interest (OFF, indicated by x symbol) (13). (B, C) Antisense RNA translational control works similarly, though antisense binding results in structures that occlude the ribosome-binding site (RBS) upstream of the gene-coding sequence to block translation (depicted by x symbols). Antisense binding can occur in a loop–loop mechanism where both antisense RNA and target mRNA are in the form of hairpin structures upon interaction (B), or a loop–linear mechanism, where either the antisense RNA or the target mRNA is unstructured (or linear) (C). (D, E) Chimeric transcription attenuators are engineered in this work by replacing portions of the S. aureus pT181 transcriptional attenuator with RNA-binding regions from natural translational regulators. (A–E) Break symbols indicate additional RNA sequence and structure not shown in the cartoons.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3753616&req=5

gkt452-F1: Antisense-RNA transcription and translation control. (A) A transcriptional attenuator that lies in the 5′ untranslated region of a transcript can fold into a structure that allows transcription (ON) when antisense RNA is not present. Antisense RNA binding to the transcribed attenuator region results in the formation of a terminator hairpin, stopping transcription before the gene of interest (OFF, indicated by x symbol) (13). (B, C) Antisense RNA translational control works similarly, though antisense binding results in structures that occlude the ribosome-binding site (RBS) upstream of the gene-coding sequence to block translation (depicted by x symbols). Antisense binding can occur in a loop–loop mechanism where both antisense RNA and target mRNA are in the form of hairpin structures upon interaction (B), or a loop–linear mechanism, where either the antisense RNA or the target mRNA is unstructured (or linear) (C). (D, E) Chimeric transcription attenuators are engineered in this work by replacing portions of the S. aureus pT181 transcriptional attenuator with RNA-binding regions from natural translational regulators. (A–E) Break symbols indicate additional RNA sequence and structure not shown in the cartoons.
Mentions: However, the ability of ncRNAs to act as fundamental building blocks of genetic circuits is only beginning to be explored. In prokaryotes, transcription attenuation offers a particularly attractive mechanism for creating RNA genetic circuitry. Transcription attenuators are ncRNAs that control the fate of transcription elongation in response to an input antisense RNA (12,13) (Figure 1A). The attenuator lies in the 5′-untranslated region of a transcript and is thought to fold into two different RNA structures during transcription that either allow (ON) or block (OFF) further elongation by RNA polymerase (12,13). An interaction with a complementary antisense RNA biases the fold to the OFF state, enabling the attenuator to act as a transcriptional switch that senses and responds to antisense RNA signals (Figure 1A). Leveraging this ability to use an RNA input to regulate an RNA output, attenuators built from the Staphylococcus aureus plasmid pT181 were recently configured in simple architectures that evaluated genetic not-or (NOR) logics, and in transcriptional cascades that propagated signals directly as antisense RNA molecules with no intermediate protein species (10).Figure 1.

Bottom Line: A comprehensive orthogonality test of these culminated in a 7 × 7 matrix of mutually orthogonal regulators.A comparison between all chimeras tested led to design principles that will facilitate further engineering of orthogonal RNA transcription regulators, and may help elucidate general principles of non-coding RNA regulation.We anticipate that our strategy will accelerate the development of even larger families of orthogonal RNA transcription regulators, and thus create breakthroughs in our ability to construct increasingly sophisticated RNA genetic circuitry.

View Article: PubMed Central - PubMed

Affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA.

ABSTRACT
Antisense RNA transcription attenuators are a key component of the synthetic biology toolbox, with their ability to serve as building blocks for both signal integration logic circuits and transcriptional cascades. However, a central challenge to building more sophisticated RNA genetic circuitry is creating larger families of orthogonal attenuators that function independently of each other. Here, we overcome this challenge by developing a modular strategy to create chimeric fusions between the engineered transcriptional attenuator from plasmid pT181 and natural antisense RNA translational regulators. Using in vivo gene expression assays in Escherichia coli, we demonstrate our ability to create chimeric attenuators by fusing sequences from five different translational regulators. Mutagenesis of these functional attenuators allowed us to create a total of 11 new chimeric attenutaors. A comprehensive orthogonality test of these culminated in a 7 × 7 matrix of mutually orthogonal regulators. A comparison between all chimeras tested led to design principles that will facilitate further engineering of orthogonal RNA transcription regulators, and may help elucidate general principles of non-coding RNA regulation. We anticipate that our strategy will accelerate the development of even larger families of orthogonal RNA transcription regulators, and thus create breakthroughs in our ability to construct increasingly sophisticated RNA genetic circuitry.

Show MeSH
Related in: MedlinePlus