Functional characterisation of RNA ligases in Escherichia coli

Recently, we have identified a novel post-transcriptional stress response mechanism in Escherichia coli. It is based on the functional specialization of ribosomes by the stress-induced endoribonucleolytic activity of MazF, the toxin component of the toxin-antitoxin system mazEF. The stress-responsive proteolysis of the labile antitoxin MazE results in the activation of MazF. Surprisingly, the endoribonuclease MazF targets the 16S rRNA in the context of the ribosome and removes the 3-terminal region harbouring the anti-Shine-Dalgarno (aSD) sequence. As base-pairing between the aSD sequence and the complementary Shine-Dalgarno (SD) sequence located on the mRNA upstream of the AUG start codon is important during canonical translation initiation, the modified ribosomes exclusively translate leaderless mRNAs that are devoid of a 5-untranslated region (UTR) and hence lack the SD sequence. As the MazF activity concomitantly leads to the generation of distinct leaderless transcripts, the translational program is modulated to adapt protein synthesis to the given conditions. Collectively, this observation raises the fundamental question about the fate of the specialized ribosomes during the recovery from stress. Considering the high complexity of the ribosome and the enormous amount of energy required for ribosome biogenesis, it is tempting to speculate that a ribosome repair mechanism might exist. This would enable the regeneration of the translational apparatus during recovery from stress without the need of the energy- and time-demanding process of de novo assembly. Thus, the main question that will be addressed in the framework of this project is the fate of the specialized ribosomes upon stress relief. Our preliminary data indicate that the truncated 16S rRNA might be repaired in the context of the ribosome by the activity of an RNA ligase. With the objective to identify the molecular mechanism underlying ribosome repair, we will in addition focus on the characterization of two annotated RNA ligases, the physiological function(s) of which still remain(s) elusive in bacteria. These studies are anticipated to shed light on key players that are involved in a completely unprecedented mechanism with respect to modulation of ribosome specificity. Hence, the results would raise the ribosome from the mere protein synthesis machinery to a regulatory hub, which can integrate environmental cues in the process of protein synthesis. Moreover, as these factors could be pivotal for pathogenic bacteria to survive and to recover from stress encountered during host infection, they could potentially represent novel targets for the design of antimicrobials, which could add on applied antibiotics.

Bacteria frequently encounter changes in environmental conditions, e.g. shifts in temperature or pH, oxidative and osmotic stress or nutrient deprivation. In particular, pathogenic bacteria are subjected to rapidly changing environments when they infect their host. To survive, bacteria have developed several strategies to adapt gene expression and protein activity to the given conditions. Our past work has shown that ribosome heterogeneity opens the possibility to promptly modify protein synthesis in response to acute environmental changes. The hallmark of this pathway is the modification of ribosomes by the stress-activated endoribonuclease MazF that targets the 16S rRNA in the context of the ribosome, thereby removing its functionally important 3'-terminus. As these modified ribosomes selectively translate a distinct pool of mRNAs likewise modified by MazF, the translational program is rapidly and energy-efficiently modulated. In light of the high complexity of ribosomes and the enormous amount of energy required for their biogenesis, in the framework of this project we addressed the question whether a ribosome repair mechanism might exist. This would enable the regeneration of the translational apparatus during stress recovery without the need of the energy- and time-demanding process of de novo assembly. Collectively, our results proved the reversibility of this 'one-step ribosome specialization' during stress relief by the activity of the RNA ligase RtcB. Employing in vitro and in vivo approaches we demonstrate that the RNA ligase RtcB catalyzes the re-ligation of the truncated rRNA present in specialized ribosomes. Thereby, their ability to translate canonical mRNAs is fully restored. Moreover, we focused on the characterization of the biological activity of RtcB besides ribosome repair. Together, our findings not only provide a physiological function for the RNA ligase RtcB in bacteria but also highlight the concept of reversible ribosome heterogeneity as a key mechanism providing a dynamic and energy-efficient alteration of protein synthesis in response to changing environmental conditions.