Tunable riboregulator switches for post-translational control of gene expression

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The switches control cellular metabolism with exquisite precision. The switches (pink) are made of RNA molecules and designed to control transmission of information (green/blue) to molecular machines (grey/orange) inside living cells.

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Living cells have multiple mechanisms to control and regulate processes—many of which involve regulating the expression of genes. Scientists have investigated ways of synthetically altering gene expression for alternative purposes, such as biosynthesis of therapeutics or chemicals. A collaboration among Laboratory experimental and theoretical biologists produced a new method to control gene expression. The key is a tunable switch made from a small non-coding RNA molecule that could have potentially useful technical applications. The Los Alamos team discovered a new modular synthetic regulatory RNA (riboregulator) class that has the potential to finely tune protein expression and independently control the concentration of each enzyme in an engineered metabolic pathway. ACS Synthetic Biology published the research.

The team developed a synthetic biology approach in which riboregulators fine-tune gene expression with potential application to metabolic engineering (Figure 2). The investigators designed the overall architecture of the riboregulators using Watson−Crick base-pairing stability. Cis-repressor (crRNA) and trans-activator RNA (taRNA) compose the riboregulators. The cisrepressor can fold to a structure that sequesters the ribosomal binding sequence, preventing translation of the downstream gene. This action blocks expression of the gene. The taRNA undergoes independent transcription. The binding and subsequent structural transition between these two regulatory RNA elements dictates whether or not the transcribed mRNA will be translated into the protein product. For example, the scientists demonstrated a cisrepressor that completely shut off translation of antibioticresistance reporters and the trans-activator that restores translation. The level of translation can be tuned based on subtle changes in the primary sequence regulatory region of the taRNA, enabling translational control of gene expression over a wide dynamic range.

The authors suggest that a targeting sequence could be modified to develop riboregulators that independently regulate translation of many genes. The investigators demonstrated that subtly changing the sequence of the trans-activator altered the ratio of the repressed and activated states and enables intermediate translational control. With a reliable method for gene regulation, scientists could potentially engineer bacteria to perform a range of important medical and industrial functions, from manufacturing pharmaceuticals to detoxifying pollutants and increasing production of biofuels.

Reference: “Tunable Riboregulator Switches for Post-transcriptional Control of Gene Expression,” ACS Synthetic Biology, Article ASAP. doi: 10.1021/acssynbio.5b00041. Authors: Malathy Krishnamurthy, Taraka Dale, Shawn R. Starkenburg, Ricardo Martí-Arbona, David T. Fox, Scott N. Twary, and Clifford J. Unkefer (Bioenergy and Biome Sciences, B-11); Scott P. Hennelly and Karissa Y. Sanbonmatsu (Theoretical Biology and Biophysics, T-6).

The Laboratory Directed Research and Development (LDRD) program funded this work, which supports the Lab’s Global Security and Energy Security mission areas and Materials for the Future science pillar. Technical contacts: Clifford Unkefer and Karissa Sanbonmatsu

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Schematic structure of the riboregulatory elements. (a) The modular design of the crRNA includes a targeting sequence (black) and a regulatory sequence (red) that forms a stem-­‐loop structure at the 5ʹ′-­‐end of the gene, sequestering the ribosomal binding sequence (RBS) and preventing translation of the messenger RNA (mRNA). (b) The taRNA is transcribed in trans and contains modules that are complementary to the targeting and variable regulatory sequences that produce stem-­‐loop structures of differential stability. The all ON version of the taRNA has little potential to form secondary structures as depicted in (d). (c) The targeting sequence binding event forms a taRNA:crRNA/mRNA complex. In this complex, translation of the target gene is either repressed or (d) the complex rearranges to free the ribosomal finding sequence and activate translation. (e) It is possible to tune the relative stability of the secondary structural elements in the crRNA and taRNA to obtain intermediate levels of translation.