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

ATPase-coupled Toprim (Topoisomerase-primase) nucleases, known as Overcoming Lysogenization Defect (OLD) proteins, are crucial for diverse antiphage defenses. The first OLD protein was discovered in phage P2 in 1970 as the factor responsible for executing P2-Lambda interference. In this classic phage conflict, P2-OLD halts phage Lambda replication in host cells carrying the P2 prophage by causing cell death through a poorly understood mechanism. We discovered P2-OLD causes cell death by degrading host threonyl-tRNA with the UGU anticodon (tRNAThrU). Phage-encoded threonyl-tRNAs with the same anticodon rescued P2-OLD-induced cell death by replacing the degraded host version. Our analysis revealed that P2-OLD cleaves tRNAs containing a paired pseudo-palindromic CNG motif in the anticodon stem, with a preference for tRNAThrU. P2-OLD cuts after the cytosine within the CNG motif on both strands, resulting in a staggered cut that detaches the anticodon stem. Phage threonyl-tRNAs resist P2-OLD cleavage due to CNG motif alterations and a shorter anticodon stem. Notably, phage tRNA repair systems cannot restore tRNAs cleaved by P2-OLD. Our findings unveil a novel tRNA inactivation mechanism involving anticodon arm amputation, providing new insights into the mechanism and specificity of Toprim nucleases and finally resolving a long-standing mystery of P2-Lambda interference.