The recent description of a new widespread class of small endonucleolytic ribozymes termed “twister” on one hand provides new opportunities for the development of advanced tools for RNA engineering and on the other hand opens essential questions on the biological utility of this and other classes of small-self cleaving motifs presenting similar distributions and genetic contexts. The present doctoral dissertation explores the potential of the twister ribozyme as a biotechnological tool for the construction of novel ribozyme-based synthetic switches and investigates its role in messenger RNA (mRNA) processing and posttranscriptional regulation in bacteria. The first part of the thesis proposes twister ribozyme as an outstandingly flexible expression platform, which in conjugation with two different aptamer domains enables the construction of different regulators of gene expression in bacteria. First, a series of one-input dependent riboswitches are generated that control gene expression at the translational level, via the sequestration of the ribosome binding site. The sensor domains (theophylline and thiamin pyrophosphate aptamers) are attached to two different sites of the catalytic core of the ribozyme using in vivo screened communication modules. Then, in vivo Boolean logic gates that act at the translational level are developed taking advantage of the fact that the twister ribozyme scaffold offers at least two independent sites for attaching ligand-sensing aptamer domains. The in vivo screening stands out as an ideal strategy to generate compact two-input riboswitches that sense and simultaneously respond to two small molecular signals and that can be described as AND, NAND, OR, NOR, ANDNOT and ORNOT binary Boolean operators. Interestingly, the described design mimics the situation observed in some naturally occurring twister ribozymes where multiple optional domains are directly connected to the catalytic core in positions P1, P3 and P5. Two-input riboswitches are also generated using a novel design in which a TPP aptamer is connected to the theophylline aptamer in an “in-series” fashion. In the second part of the dissertation, several bacterial twister motifs, previously identified in intergenic regions and presenting different configurations and structural organizations, are characterized using both in vitro and in vivo assays. The analysis of their genetic contexts shows that these motifs have the potential of being transcribed as part of polycistronic mRNAs, thus we suggest the involvement of bacterial twister motifs in the processing of mRNA. Our data show that the ribozyme-mediated cleavage of the bacterial 3’- untranslated region (3’-UTR) has major effects on gene expression. While the observed effects correlate weakly with the kinetic parameters of the ribozymes, they show to depend primarily on the motif-specific structural features and on mRNA stabilization properties of the secondary structures that remain on the 3’-UTR after ribozyme cleavage. Using these principles, novel artificial twister-based riboswitches are developed that exert their switching activity via ligand-dependent cleavage of the 3’-UTR and the removal of the protective intrinsic terminator stem-loop. The fact that these artificially developed switches can effectively regulate gene expression relying on the cleavage of the 3’-UTR suggests that similar ligand-dependent modulation of the ribozyme activity could exist also in nature with possible functions in gene expression regulation. Overall, this dissertation describes the experimental procedures and design approach to employ the twister ribozyme for the construction of new tools that have the potential of being broadly applied in biotechnology, synthetic biology and metabolic engineering. The evidences deriving from the characterization of a number of bacterial twister ribozymes and the observed versatility in our artificial gene-regulatory setups are used to discuss possible natural roles of this newly discovered and widespread catalytically active motif in bacteria.publishe
