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Characterization of natural and synthetic riboswitches using gel mobility shift assays.
Riboswitches are cis-acting RNA elements that alter gene expression in response to small ligands. The ligand in turn regulates gene expression through a ligand-dependent interaction with the riboswitch. Riboswitches have been identified in several unrelated bacteria, all of which appear to respond to specific ligands. This suggests that riboswitches may have similar structures. Here we present a comparison of the structures and sequences of the five riboswitches that have been identified in Escherichia coli. We have isolated the riboswitches from natural sources and found that they are comprised of three RNA elements, called leader sequence, aptamer, and terminator, that are related to but distinct from the sequence motifs found in other known natural regulatory RNAs. We have produced a synthetic riboswitch that is similar to the naturally occurring riboswitch that responds to the ligand cytidine in the yeast Saccharomyces cerevisiae. We have also shown that the E. coli riboswitch, and its synthetic counterpart, form specific, stable complexes with ligand. In particular, cytidine-binding riboswitches respond to cytidine by an increase in the mobility of the aptamer-ligand complex in native polyacrylamide gels. Because this reaction is specific, gel shift assays can be used to characterize these RNAs, and for the first time, identify ligand-binding specificities in natural riboswitches.Q:
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