Considerations for Using Hammerhead-Based Riboswitches in the 5′-UTR to Control Genes in Bacteria

Riboswitches are small regulatory elements composed of RNA that have garnered much attention over the past decade due to their high selectivity. These single-stranded nucleic cids are generally composed of two functional domains: (i) an aptamer domain, which selectively binds a specific target molecule, and (ii) a functional domain or expression platform that controls whether the gene is turned “on” or “off” through allosteric means. The majority of natural riboswitches are located upstream of the genes they control, typically repressing expression of the genes they control when the ligand they recognize reaches a specific concentration. However, many synthetic riboswitches have been constructed that function in either the 5′-UTR or the 3′-UTR, depending on their mechanism.

 The hammerhead ribozyme has been used as a riboswitch functional domain for ligand-dependent control of gene expression by linking an aptamer to one of its three arms. The most successful use of a hammerhead riboswitch thus constructed has been demonstrated in the 3′-UTR of eukaryotic organisms. In prokaryotic organisms, the 3′-UTR switch does not control gene expression well, and 5′-UTR switches have met with limited success. Here, we investigate various design considerations, such as use of an anti-RBS fragment upstream of the riboswitch, number of nucleotides between the riboswitch and gene, and speed of protein synthesis, to use hammerhead riboswitches placed in the 5′-UTR to control expression of the lacZ and gfp genes in the bacterium Escherichia coli. The results of this work will provide a groundwork for design of efficient 5′-UTR hammerhead riboswitches in prokaryotic organisms.