OLD amputates the anticodon arm of tRNAs during P2-Lambda interference

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• ATPase-coupled Toprim (Topoisomerase-primase) nucleases, known as Overcoming Lysogenization Defect (OLD) proteins, are vital for antiphage defenses.
• P2-OLD protein, discovered in 1970, mediates P2-Lambda phage interference by halting Lambda replication in P2 prophage-carrying cells.

Purpose of the Study:

• To elucidate the mechanism by which P2-OLD protein induces cell death.
• To identify the specific molecular target of P2-OLD.
• To understand the basis of P2-Lambda interference.

Main Methods:

• Biochemical assays to identify P2-OLD targets.
• RNA sequencing and analysis to characterize cleaved tRNAs.
• Structural analysis of tRNA motifs recognized by P2-OLD.

Main Results:

• P2-OLD specifically degrades host threonyl-tRNA with the UGU anticodon (tRNAThrU).
• Cleavage occurs at a pseudo-palindromic CNG motif in the anticodon stem, resulting in anticodon arm amputation.
• Phage threonyl-tRNAs resist cleavage due to altered CNG motifs and shorter anticodon stems, while host repair systems cannot restore cleaved tRNAs.

Conclusions:

• P2-OLD employs a novel tRNA inactivation mechanism involving anticodon arm amputation.
• This mechanism explains P2-Lambda interference and provides insights into Toprim nuclease specificity.
• The findings resolve a long-standing mystery in phage-host interactions.