Objective:
Bacteriophages, viruses that infect bacteria, often encode transfer RNAs (tRNAs) with sequences that differ substantially from those of their bacterial hosts; however, their specific role (s) in enhancing phage replication remain unclear. This study investigates the function of phage-encoded initiator tRNAs along with their epitranscriptomic RNA modification landscape.
Methods:
Phage initiator tRNAs were expressed in E. coli from plasmids, and host growth, plasmid stability, and translation initiation efficiency were evaluated. An LC–MS/MS-based tRNA sequencing workflow was used to identify modifications in bacterial-produced tRNAs, with in vitro-transcribed tRNAs as unmodified controls.
Results:
Phage initiator tRNA expression in E. coli BL21 was stable and did not impact growth, suggesting no inherent toxicity in this genetic background. An LC–MS/MS workflow was developed, and both in vitro transcribed standards and bacterial-produced tRNAs were measured, with several epitranscriptomic modifications identified.
Conclusion:
This study creates an analytical framework to characterise the functional activity and epitranscriptomic landscape of phage-encoded tRNAs. These methods advance our current understanding of the roles and modification landscape of phage-encoded tRNAs, with the overall aim of defining their interactions with the host translational machinery and antiphage defence systems. These insights will aid future efforts to select and engineer phage with improved translational efficiency and therapeutic potential.