Alex’s paper “Achieving large dynamic range control of gene expression with a compact RNA transcription-translation regulator” is online in Nucleic Acids Research. In this work we report a breakthrough in RNA engineering that allowed us to create some of the largest dynamic range RNA regulators we currently know of that can be used to solve major challenges in the construction of synthetic RNA circuitry.
A long-standing goal of synthetic biology is to develop molecular building blocks that can be used to design genetic networks that can reliably control cell behavior. Increasingly, synthetic biologists are turning to RNA regulatory mechanisms for their versatility, our ability to predict and design their structures and function, and their desirable dynamic properties compared to similar protein regulators like being able to propagate signals quickly. However, many RNA regulatory mechanisms suffer from poor dynamic range – i.e. they often display leaky behavior that can be incorrectly propagated throughout a network.
We’ve seen this problem in previous work in building RNA circuits out of the pT181 transcriptional attenuator system. The pT181 mechanism is an RNA switch that controls transcription – by itself it forms into an RNA structure that allows transcription to proceed, but when a special complementary RNA called an antisense RNA is present the switch is flipped to stop transcription. The specific RNA structure that actually forms to stop transcription is called a transcription terminator – it’s a special hairpin that forms next to a polymerase that is stalled at a polyU sequence and causes the polymerase to stop transcription. While the pT181 system has been useful for building all sorts of RNA genetic circuitry, it has an issue with leak – even when antisense RNA is present the switch is only about 85% effective at stopping transcription.
We’ve been mulling this problem for a while and during that process we actually noticed that the pT181 terminator itself contains a ribosome binding sequence. This was a pretty interesting surprise and it gave us a clue that maybe the system was controlling both transcription and translation. If this was true then this might be a way to fix leak by controlling both aspects of gene expression.
So we launched into an investigation of the ‘dual control’ system which is reported here. The dual control pT181 attenuator regulates gene expressing using transcriptional termination and translational RBS occlusion to increase the dynamic range of the pT181 repressor and an activating version of this mechanism called a STAR. The cool thing was that it works spectacularly! Our dual transcription-translation control regulators can achieve dynamic ranges of 50 fold repression and greater than 900 fold activation – the largest fold activation of any RNA regulatory mechanism shown to date! Part of the reason we thinks this works so well is the redundant level of genetic control – if any RNA polymerase accidentally escapes the transcriptional terminator hairpin, translation is still prevented because the ribosome binding site is bound into the hairpin. We also found that the ON levels are increased – likely because in the RNA structure of the ON state the ribosome binding site is even more exposed by being next to a strong RNA structure. Interestingly we also noticed this feature in the study of a natural riboswitch, suggesting that this dual control strategy could be exploited throughout nature to offer tighter regulation for RNA mechanisms. Or more speculatively, could represent some evolutionary middle point between the evolution of transcriptional and translational control by RNA hairpin structures.
In this work we also showed that they work in multiple genetic contexts including regulating single genes and multi-gene operons. We can also create libraries of dual control repressors that can function independently of each other by extending the method to additional orthogonal transcriptional regulators. These new riboregulators can be integrated into the bottom level of a transcriptional RNA cascade to reduce network leak. Moving forward, we hope to use these regulators to build more sophisticated networks with complex regulatory functions.
Overall this is another example of how RNAs configured correctly can work just as well as protein based regulators and we hope this inspires more innovations in RNA synthetic biology!