184 9 Small Molecule-Responsive RNA Switches (Bacteria)
9.3.2 Transcriptional Regulation
Relative to translationally regulated riboswitches, transcriptional regulation has
not been extensively exploited in engineered riboswitches. Although this could
be partly due to the complex mechanisms observed in the transcriptionally regu-
lated natural riboswitches that involve folding kinetics of multiple RNA elements
[25], several recent publications indicate that it is possible to engineer such
riboswitches. Wachsmuth et al. used computational tools to rationally design a
transcriptionally regulated riboswitch using a theophylline aptamer [20]. After
some iterative improvements, they obtained a riboswitch with a respectable ON/
OFF ratio of 6.5 in E. coli.
Alternatively, Qi and coworkers engineered trans‐acting small noncoding
RNAs (ncRNA) that function by transcriptionally regulating the target gene
[26]. Their system is based on the antisense RNA‐mediated transcription
attenuation observed in the staphylococcal plasmid pT181 [27, 28]. By stra-
tegically fusing an aptamer and the ncRNA in tandem and screening of
mutants in E. coli, the group successfully isolated small molecule‐regulated
trans‐ acting RNA switches.
More recently, Ceres and coworkers discovered that certain expression plat-
forms of transcriptionally regulated riboswitches can accommodate different
natural and synthetic aptamers without losing the gene regulatory function [29].
They were also able to qualitatively tune the device characteristics by adjusting
the strength of the key stem sequences in a predictable fashion.ON OFFOFF ONRBS(a)(b)LigandNN...NN3 ′5 ′Ligand RBSLigandLigandNN...NN3 ′5 ′RBSN Cleavage
N
NN
N
NN
N
NN
N
3 ′ N5 ′RBS3 ′5 ′5 ′Figure 9.1 (a,b) Examples of synthetic riboswitch libraries.