RNA is arguably the most functionally diverse biological macromolecule. In some cases a single discrete RNA sequence performs multiple roles and this can be conferred by a complex three-dimensional structure. This multifunctionality can also be driven or enhanced by the ability of a given RNA to assume different conformational (and therefore functional) states1. Despite its biological importance, a detailed structural understanding of the paradigm of RNA structure-driven multifunctionality is lacking. Examples to address this gap are found in single-stranded positive-sense RNA viruses, a prototype being the tRNA-like structure (TLS) found at the 3′ end of the Turnip Yellow Mosaic Virus (TYMV). This TLS not only acts like a tRNA to drive aminoacylation of the viral genomic RNA (gRNA)2-4, but also interacts with other structures in the gRNA's 3′ untranslated region5, contains the promoter for negative strand synthesis, and influences several infection-critical processes6. This TLS RNA can provide a glimpse into the structural basis of RNA multifunctionality and plasticity, but for decades its high-resolution structure has remained elusive. Here, we present the crystal structure of the complete TYMV TLS to 2.0 Å resolution. Globally, the RNA adopts a shape that mimics tRNA, but it uses a very different set of intramolecular interactions to achieve this shape. These interactions also allow the TLS to readily switch conformations. In addition, the TLS structure is ‘two-faced’: one ‘face’ closely mimics tRNA and drives aminoacylation, the other ‘face’ diverges from tRNA and enables additional functionality. The TLS is thus structured to perform several functions and interact with diverse binding partners, and we demonstrate its ability to specifically bind to ribosomes.

The central dogma of gene expression (DNA→RNA→protein) is universal, but in different domains of life there are fundamental mechanistic differences within this pathway. For example, the canonical molecular signals used to initiate protein synthesis in bacteria and eukaryotes are mutually exclusive1,2. However, the core structures and conformational dynamics of ribosomes that are responsible for the steps of translation following initiation are ancient and conserved across the domains of life3,4. We asked whether an undiscovered RNA-based signal might be able to use these conserved features, bypassing mechanisms specific to each domain of life, and initiate protein synthesis in both bacteria and eukaryotes. Although structured internal ribosome entry site (IRES) RNAs can manipulate ribosomes to initiate translation in eukaryotic cells, an analogous RNA structure-based mechanism has not been observed in bacteria. Here, we report our discovery that a eukaryotic viral IRES can initiate translation in live bacteria. We solved the crystal structure of this IRES bound to a bacterial ribosome to 3.8 Å resolution, revealing that despite differences between bacterial and eukaryotic ribosomes this IRES binds directly to both and occupies the space normally used by tRNAs. Initiation in both bacteria and eukaryotes depends on the structure of the IRES RNA but in bacteria this RNA uses a different mechanism that includes a form of ribosome repositioning after initial recruitment. This IRES RNA bridges billions of years of evolutionary divergence as an example of an RNA structure-based translation initiation signal capable of operating in two domains of life.