| Abstract [eng] |
Argonaute (Ago) proteins, the key components of RNA interference (RNAi) machinery, are ubiquitous across all domains of life. While eukaryotic Argonautes (eAgos) have been extensively studied, prokaryotic Argonautes (pAgos) exhibit greater structural and mechanistic diversity. This doctoral thesis investigates a truncated long-B pAgo from Archaeoglobus fulgidus (AfAgo) and its associated protein, AfAgo-N, aiming to elucidate their structural and biochemical characteristics. AfAgo is classified as a long-B type despite comprising only the MID and PIWI domains, resembling short pAgos. The research explores the oligomeric state of AfAgo in vitro, revealing its ability to form homodimeric complexes and looped dsDNA structures, a feature not previously observed among pAgos. Furthermore, AfAgo displays specificity for the 5′-terminal AUU sequence of the guide RNA and demonstrates RNA-guided ssDNA targeting in vitro, characteristics not previously described for this protein, expanding our understanding of pAgo functionalities. Moreover, a novel protein, AfAgo-N, is identified within the same operon as AfAgo in A. fulgidus. Structural analysis reveals the equivalence of AfAgo-N to the N-L1-L2 domains of long pAgos. It forms a heterodimeric complex with AfAgo, termed fAfAgo, via the same dimerization surface used by AfAgo to form homodimers. fAfAgo exhibits RNA-guided DNA targeting activity and displays enhanced affinity for DNA targets compared to stand-alone AfAgo. These findings significantly broaden our understanding of pAgo functionalities, unveiling previously unexplored features such as homodimerization and sequence specificity beyond the first 5′-nucleotide. The discovery of fAfAgo, a heterodimeric PAZ-less pAgo, highlights the complexity of pAgo-associated protein interactions. This comprehensive characterization of AfAgo and AfAgo-N lays the groundwork for future research into their possible roles in defense mechanisms or other functions and paves the way to investigate other possible split pAgo systems. |
