| Abstract [eng] |
Bacteria and archaea use CRISPR-Cas systems to defend themselves against viral infection. These adaptive immune mechanisms detect and neutralize invading nucleic acids. Among them, type III CRISPR-Cas systems are unique: alongside viral RNA degradation, they trigger an additional layer of defense by producing signaling molecules, cyclic oligoadenylates (cAn), which activate various downstream effectors. This thesis explores two of these effectors: the ribonuclease Csm6 and a Lon-SAVED fusion protein CalpL of the tripartite CalpL-CalpT-CalpS effector. Csm6 is activated by cA6 binding to its CARF domain, triggering HEPN domain-mediated RNA degradation. Cell metabolite analysis and biochemical assays revealed that Csm6 also degrades its own activator, cA6, creating a built-in self-limiting loop. The second part characterizes the CalpL-CalpT-CalpS effector, which responds to cA4. Biochemical and in vivo toxicity assays showed that cA4 binding activates CalpL to cleave the anti-σ factor CalpT, marking it for degradation by cellular proteases and thereby releasing the σ factor CalpS. CalpL also degrades its own activator, functioning as a self-limiting effector. Furthermore, structural studies revealed how filament formation governs CalpL’s protease and the regulatory ring nuclease activities. Together, these findings show how type III effectors integrate cAn signaling with intrinsic regulatory mechanisms. |
