If you can pronounce CRISPR, you know RNA-mediated gene control has been a rising star in synthetic biology. In principle, RNA regulation allows gene circuits to have many more independent control switches than traditional protein transcription factors. CRISPR has turned out to be a great system for RNA-guided gene repression, but it hasn’t been as good for activation in bacteria. Fortunately, a new RNA-guided acronym has arrived to fill the inky black transcriptional activation void: small transcription activating RNAs (STARs).
How STARs Work: The Terminator Analogy
STARs work by targeting terminators, which are transcribed sequences that form strong RNA stem-loops followed by long poly-U tracts. Like an Austrian cyborg assassin sent from the future, the strong hairpins terminate transcription by yanking the poly-U tracts out of the RNA polymerase. The first terminator targeted for activation was model pT181, which is naturally preceded by a complementary anti-terminator sequence.
Continuing our analogy, this system also has an ‘anti-terminator’ or rebel freedom fighter sent from the future, which base pairs with the 5′ end of the terminator, preventing termination. This particular model of terminator also comes standard with an anti-anti-terminator (aka. attenuator), a small RNA transcribed in trans that base-pairs with the anti-terminator, preventing it, like a backfiring homemade pipe bomb, from stopping the terminator (if you’re counting, 3 negatives = repression).
Turning Repression into Activation
To make a small RNA that instead activates transcription, researchers led by Julius B Lucks first placed the anti-anti-terminator in front of the anti-terminator, and then expressed yet another complementary RNA, an anti-anti-anti-terminator, in trans. Like a conveniently located hydraulic press, the anti-anti-anti-terminator finally stopped the terminator, activating the downstream GFP by up to 11-fold.
Providing respite to our overextended allusion, the next step for the researchers was to remove two layers of anti’s, designing STAR anti-terminators that directly target native E. coli terminators. These achieved orthogonal activation ranging from 3- to 94-fold. Longer STARs weren’t necessarily better; their anti-termination efficiency seemed to depend on intra-molecular secondary structure.
To demonstrate the power of their new STARs, the authors built RNA-only transcriptional AND and A-and-not-B gates, both beyond the reach of repression-only CRISPR systems. Anti-terminating STARs still require some trial and error to achieve strong activation, but they extend RNA regulation to include transcriptional activation. It will be interesting to see if there is a sequel.
Don’t terminate here! Read more about this stellar work in Nature Chemical Biology, February 2015.