Conditional gene expression using ribozymes : Post-transcriptional control of amino acid identity in protein synthesis and temperature-dependent gene expression

During the main research study we wanted to demonstrate that it is possible to control the amino acid identity in vivo on a post-transcriptional level by switching designer tRNAs via the ligand-dependent regulation of ribozyme activity.For this purpose we designed and generated various modular constructs that are composed of distinct parts: an aptamer, a catalytic ribozyme core (hammerhead ribozyme) and of course the appropriate amber suppressor tRNA. Our design included the connection of aptamer (theophylline or thiamine pyrophosphate) to ribozyme via a small connecting sequence, which was randomized. With the help of in vivo screening process we managed to identify functional riboswitches that possess a desired activity (ON or OFF). This activity resulted in the conditional release or not of a suppressor tRNA which in turn serves as tool used for decoding of the amber stop codon in the mRNA of our reporter gene, a fluorescent protein.We managed to obtain novel, individual tRNA riboswitches that we successfully combined in a dual fashion, in order to switch one or the other tRNA, therefore the protein synthesis of one or the other protein variant.Considering the fact that suppression efficiency is inherently low due to release factor competition to the generated suppressor tRNA, this methodology is not quantitative. However, we demonstrated that the protein of interest can be selectively generated in relatively high yields with the use of desired small molecule trigger.This idea can be further developed and exploited in ways that benefit the field of protein chemistry. Considering the high modularity of our constructs, one can envision the multitude of possibilities arising from substituting or expanding by adding one or more parts to the construct. For example, use of different aptamer sensor in combination with a tRNA will unlock the potential generation of more than two different protein variants, using all the while, the same message RNA containing one amber stop codon for which all suppressor tRNAs compete for. Of course optimization of the current system by utilizing more stringent expression control systems could improve the overall performance of the riboswitching system. Another approach which can enhance the performance of our current system is the use of release factor knockout strains which is documented to double the efficiency of suppression systems. Moreover, the applicability of our already versatile tool can be exponentially increased when combined with the fascinating and thriving field of unnatural amino acids. Considering the fact that the central dogma is highly conserved throughout all domains of life, our approach should be able to be realized in higher organisms as well.Finally, we demonstrated that it is possible to design and synthesize ribozyme-based gene regulation elements that are able to respond to temperature changes. This can be achieved with the aid of a thermosensing hairpin that melts with increased temperatures which in turn modulates the function of the hammerhead ribozyme. By replacing the ligand responsive aptamer in the hammerhead ribozyme construct with a Salmonella RNA thermometer we expanded the concept of hammerhead ribozyme regulated gene expression. In this new fusion construct the hammerhead ribozyme functions by exposing a ribosome-binding site upon self-cleavage, while an RNA hairpin undergoes a conformational change caused by an increase in temperature. After in vivo screening of several temperature-sensitive clones for gene regulation activity, we identified two of such thermozymes. The designed tool acts in reverse manner to the natural thermo-sensitive RNA hairpin, which is used by Salmonella to increase gene expression. New tools were developed in order to regulate gene expression in response to temperature changes.On the whole, we presented methodologies that include the use of RNA-only toolboxes which can be adapted, modified and applied in the field of protein chemistry and protein engineering. Further development of the existing tools, along with their potential application in higher organisms, is possible to shed light in investigation of protein synthesis pathways, mutant generation and their mechanisms and functions