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Design of artificial, ribozyme-based genetic switches in bacteria

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WIELAND, Markus, 2010. Design of artificial, ribozyme-based genetic switches in bacteria [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Wieland2010Desig-9677, title={Design of artificial, ribozyme-based genetic switches in bacteria}, year={2010}, author={Wieland, Markus}, address={Konstanz}, school={Universität Konstanz} }

Wieland, Markus Design of artificial, ribozyme-based genetic switches in bacteria Wieland, Markus The aim of this PhD thesis was the design and construction of Hammerhead ribozyme-based riboswitches that control gene expression in E. coli.<br />At the beginning of this work, such switches were already developed for eukaryotic cells by inserting the HHR into the non-coding sequences of a reporter mRNA. HHR self-cleavage detached the essential 5 -cap or 3 -poly(A) tail from the mRNA thus severely decreasing mRNA stability and eventually gene expression levels. Since the genetic machinery of E. coli differs strongly, a simple transfer of this way of action was not feasible. In deed, it was necessary to conceive a novel and sophisticated mechanism for the HHR to effect reporter gene expression.<br />The starting point of this work was to implement the HHR into a bacterial mRNA coding for the reporter gene. Inspired by the natural occurring riboswitches, we aimed for regulation of translation initiation by sequestering / liberating the accessibility of the ribosome binding site (RBS). In order to achieve that, we incorporated the RBS in an extended stem I of the HHR scaffold thereby inhibiting ribosome binding. Only upon self-cleavage is the RBS liberated and reporter gene expression is induced. This HHR setup is highly advantageous for its use in bacteria: On the one hand, it allows the formation of tertiary interactions between ribozyme stem I and II which are essential for effective cleavage in vivo. On the other hand, stem III sequence is completely variable thus presenting a promising position for attaching aptamer domains in order to externally regulate ribozyme activity and eventually gene expression levels.<br />A major task in the construction of such allosteric ribozymes has always been the optimization of the connection sequence between the two functional domains. For the construction of aptamer-regulated HHRs (HHAz) in vivo, it can not be taken for granted that aptazymes optimized in vitro can be transferred in living beings although it was successfully performed in another attempt. Therefore we established an in vivo screening method in order to directly identify relevant connection sequences in the context where they are actually used. Applying this method we constructed a theophylline-dependent aptazyme which was capable of inducing gene expression almost ten fold after ligand addition to the growth medium. For the first time, this setup provided the opportunity to use an aptazyme for bacterial gene expression control.<br />Based on this mechanistic design, we exchanged the artificially derived theophylline aptamer sequence with the TPP aptamer domain from the corresponding natural riboswitch. After another in vivo screening, we identified several clones inducing as well as inhibiting gene expression. The simple but effective exchange of the aptamer domains in these switches is an impressive proof for the generalizable use of RNA domains. It would be very interesting to investigate if other natural aptamer domains could be used in this setup, too.<br />By attaching an additional fourth helical region to the HHR scaffold and its subsequent implementation in the bacterial mRNA context, we were able to introduce a further insertion site for an aptamer domain. As a proof of concept, we attached the theophylline aptamer to this fourth site, performed an in vivo screening and eventually identified one variant which showed theophylline-dependent induction of ribozyme activity and eventually eGFP expression. This additional attachment-site could be very valuable for the construction of logic gates based on an HHR with two simultaneously attached aptamers in E. coli. Furthermore, the expanded HHR scaffold enables its use as a gene silencer acting in trans by cleaving a recognition site on an mRNA without interfering with the formation of the catalytically important tertiary interactions between stem I and II. This is currently pursued in collaboration with the laboratory of Prof. Dr. Citti, Italy. Noteworthy, the design of the expanded HHR scaffold was strongly inspired by natural three-way junctions thereby again demonstrating the general combinability of certain RNA elements with each other.<br />Besides aptazyme-mediated regulation of translation initiation, we also used the HHR to control tRNA and splicing activity as well as ribosome stability.<br />For the regulation of tRNA activity, we conceived a comparable gain of function mechanism as used in the mRNA context: We integrated essential elements of the tRNA in stem I of the HHR thereby destroying the typical tRNA cloverleaf structure. The new fold is not recognized anymore by essential tRNA processing enzymes leaving this tRNA to be inoperative in translation. By self-cleavage, however, the original tRNA fold is rescued and the tRNA can be eventually used in translation. Ligand-controlled tRNA activity was achieved by transferring the mRNA-based theophylline-dependent aptazyme to this new setup hence again proofing the generalizable use of RNA elements once identified.<br />In order to render ribosome stability small molecule-dependent, we simply inserted a TPP-dependent aptazyme in helix 6 of the 16 S rRNA. Interestingly, the HHR scaffold inserted at this position did not interfere with ribosome activity in vivo, while self-cleavage almost completely inactivated it. By an in vivo screening, we identified once more TPP-dependent aptazyme variants which are either inducing or inhibiting catalytic activity. One of the identified variants featured the identical connection sequence as a variant origination from the mRNA context. Again, this is a very impressive example for the compatibility of RNA domains inserted in different surroundings as well as a validation for the screening method itself. In future, these aptazyme-dependent ribosome variants could be used to construct artificial gene networks in which expression levels of several genes have to be controlled simultaneously. Moreover, it might represent an interesting tool for in vitro and in vivo characterization of 16 S rRNA folding.<br />Finally, we were also able to control group I splicing activity in E. coli in a comparable way as already shown with the 16 S rRNA by inserting the HHR into helix 10 of the Tetrahymena thermophila intron. While the ribozyme scaffold itself did not interfere with splicing activity, HHR self-cleavage had a strong effect on splicing-activity.<br />As a conclusion of this thesis, we have shown that the Hammerhead ribozyme represents a very powerful and versatile tool for the artificial regulation of gene expression in E. coli. The constructed switches follow a rationally-designed and protein-independent mechanism and the developed RNA parts are highly interchangeable between several surroundings (mRNA, tRNA, and rRNA). Moreover, it has been show that at least the theophylline-dependent HHAz can be also transferred to eukaryotic systems while retaining its switching properties (Master thesis of Patrick Ketzer and Simon Ausländer and).<br />For synthetic biology, it would be very appealing to combine several of these switches in order to construct genetic circuits. Our lab has recently shown, that it is possible to combine several of the here presented switches in one system in order to design complex logic gates. Considering the small size of ribozyme-based compared to the protein-based switches, the use of the RNA parts might be advantageous. deu application/pdf 2011-03-24T18:13:38Z terms-of-use 2011-03-24T18:13:38Z 2010

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