tRNA as an assembly chaperone for a macromolecular transcription-processing complex

tRNAs are widely recognized for their role in translation. Here we describe a previously unidentified function of tRNA as an assembly chaperone. During poxviral infection tRNAGln/Arg lacking the anticodon mcm5s2U modification is specifically recruited from the cellular tRNA pool to a multi-subunit poxviral RNA polymerase complex (vRNAP), where it controls the transition to the pre-initiation complex upon infection. Cryo-EM analysis of assembly intermediates illustrates how tRNAGln/Arg orchestrates the recruitment of transcription and mRNA processing factors to vRNAP. The atomic vRNAP structures reveal an induced-fit mechanism that internalizes anticodon base G36 into the anticodon stem creating a non-canonical tRNA structure and selecting a defined tRNA modification pattern. The role of tRNA as assembly chaperone extends to the pathogenic MPXV due to the high conservation of vRNAP.