Cryo-Electron Microscopy (Cryo-EM) Structure of a Cap-Independent Translational Enhancer of the Turnip Crinkle Virus (TCV) Bound to the Eukaryotic Ribosome

Many plant viruses, unlike animal viruses, use 3′ translational enhancers that function by unknown mechanism to achieve translation initiation. A translational enhancer in the 3′-UTR of turnip crinkle virus (TCV) has been shown to synergistically enhance translation when associated with the TCV 5′-UTR. The major enhancement has been shown, biochemically, due to a T-shaped structure (TSS) which binds to the 80S ribosomes with the aid of a pseudoknot that mimics the tRNA acceptor stem. The research presented here focuses on the structural interaction of the TSS structure with the 80S eukaryotic ribosome from Saccharomyces cerevisiae using the single-particle cryo-EM reconstruction technique. Our preliminary work shows that the density corresponding to the TSS structure is directly visible in the cryo-EM map, and most of the ribosomes containing the TSS structure are in the ratcheted-like intersubunit-rotated state. These results match the biochemical results. We are currently investigating the detailed binding interactions between the TSS and the ribosome. Interpretation of this cryo-EM map is a first step toward an understanding of the molecular mechanism of translation enhancement and regulation by the TSS structure.