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and reported translation inhibition ranging from 0 to 90%. These data were used
to build a quantitative model that was able to explain 87% of the variability of in vivo
performance of the RNA regulators. The authors then demonstrated the utility of this
model by forward-engineering new RNA regulators, as well as families of up to six
mutually orthogonal regulators. More interestingly, the quantitative model described
enables designers to explore regulators that vary across a continuous range of regulation.
To add sensing functions to translational regulators, one may fuse aptamer domains to
RNA control parts. For example, Qi et al. utilized their aptamer grafting strategy (described
above for pT181) to also create IS10 variants that responded to protein and small molecule
inputs. 34 This effectively creates artificial riboswitches. These can also be accessed by taking
natural riboswitches and changing their specificity for their cognate ligand. For example,
Dixon et al. reported the construction of synthetic variants of the add A-riboswitch that
respond not to the natural ligand adenine, but to related heterocyclic analogues. 40 The add
A-riboswitch sequesters an embedded RBS in the OFF state but releases the RBS to create the
ON state when its cognate adenine ligand, recognized by a handful of hydrogen-bonding
contacts, is present. To make variants of this translational regulatory riboswitch, the authors
first synthesized a small library of mutants randomized in the ligand-binding site. These
mutants were then attached to control chloramphenicol acetyl transferase (CAT) expression
and screened in vivo for translational activation of CAT in the presence and absence of a
collection of heterocyclic compounds. This screen resulted in a number of riboswitches
specifically activated by nonnative ligands that can now be used as inducers of translational
activation. In a more recent study, one riboswitch variant triggered by ammeline was used
to construct a mutually orthogonal riboswitch for the inducible independent expression of
two genes under the control of the two orthogonal riboswitches. 41
As in transcription, the emerging theme is to create layers of orthogonal but similarly acting,
sensing and regulatory elements derived from common parents such that relatively simple
models of quantitative function, derived from sequence, may be obtained. This enables a
designer to more rationally search a
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parameter
space rather than a sequence space to
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achieve a desired target.
Degradation
For an mRNA to be translated, it must be stable inside the cell. Therefore, the stability of an
mRNA transcript is also a key determinant of gene expression. Parts that control mRNA
stability have thus been constructed.
For example, Babiskin and Smolke sought to extend the regulatory tools to control gene
expression in eukaryotic organisms by engineering an endogenous RNA control system that
regulates the stability of mRNAs. 11 In eukaryotic cells, endoribonucleases can cleave mRNA
transcripts and induce their rapid degradation by exoribonucleases. In Saccharomyces
cerevisiae , the RNase III enzyme, Rnt1p, recognizes specific RNA hairpins and cleaves its
substrates. The authors therefore used a screening strategy to generate Rnt1p substrate
variants that exhibited variable cleavage activity and consequently, a wide range of gene
expression (between 8% and 85%). They also examined the modularity of their devices by
verifying it would perform reliably with two sequence divergent fluorescent reporters. The
resulting library is a set of post-transcriptional RNA-based controllers that can be inserted
into the 3
-UTR of any transcript to yield predictable levels of gene expression.
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Degradation-based regulatory parts that sense inputs have also been created. For example, in
a number of systems developed by the Smolke laboratory, the addition of engineered
ribozymes in the 3
-UTR of yeast mRNA transcripts allows the regulated cleavage of poly-A
tails, which are required for the proper export, translation, and stability of mRNAs. 42 By
linking ribozymes to aptamer domains in various configurations, Smolke has demonstrated
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