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

Transfer RNAs (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, tRNA Gln/Arg lacking the anticodon mcm 5 s 2 U34 modification is specifically sequestered from the cellular tRNA pool to promote formation of a multisubunit poxviral RNA polymerase complex (vRNAP). Cryo-electron microscopy analysis of assembly intermediates illustrates how tRNA Gln/Arg orchestrates the recruitment of transcription and mRNA processing factors to vRNAP where it controls the transition to the preinitiation complex. This is achieved by an induced fit mechanism that internalizes anticodon base G36 into the anticodon stem, creating a noncanonical tRNA structure and selecting a defined tRNA modification pattern. The role of tRNA as an assembly chaperone extends to the pathogenic Mpox virus, which features a similar vRNAP.