(530f) Regulation of Gene Expression By the Twister Sister Ribozyme

Riboswitches are structured non-coding RNA molecules that provide the ability to control gene expression within a coding sequence. Several ribozyme-based riboswitches have been constructed over the past decade by linking a ligand-responsive aptamer to a ribozyme. The twister sister motif represents a newly discovered class of ribozyme closely resembling the previously characterized twister ribozyme. Like the twister ribozyme, the twister sister ribozyme was found to demonstrate self-cleaving behavior in vitro. Furthermore, the twister sister ribozyme expresses self-cleaving activity both faster and with less co-factor magnesium cost than other riboswitch candidates. Thus, the goal of the project is to develop synthetic riboswitches by employing the twister sister ribozyme as a functional domain.

To begin, initial characterization was performed to determine if the twister sister ribozyme could be used to control gene expression in vivo. We integrated the twister sister ribozyme through each of the four different stems upstream of the gfp gene in an E. coli expression plasmid, creating four different plasmids. In vitro transcription using the plasmids as a template revealed that all four ribozyme constructs produce mRNA that is capable of self-cleavage, indicating that the ribozyme may serve as a useful functional domain for riboswitch activity in vivo. Based upon these results, the plasmids were transformed into BL21(DE3), and fluorescence of each strain was measured, indicating the ability of the twister sister ribozymes to regulate gene expression in E. coli. Two ribozyme candidates successfully regulated expression of gfp in E. coli in a cleavage-dependent fashion. Future work will include attaching different ligand-binding aptamers to the ribozyme to develop and investigating a more flexible ligand-inducible gene-regulating riboswitch system.