Cryo-EM structure of the fully assembled Elongator complex

Abstract

Transfer RNA (tRNA) molecules are essential to decode messenger RNA codons during protein synthesis. All known tRNAs are heavily modified at multiple positions through post-transcriptional addition of chemical groups. Modifications in the tRNA anticodons are directly influencing ribosome decoding and dynamics during translation elongation and are crucial for maintaining proteome integrity. In eukaryotes, wobble uridines are modified by Elongator, a large and highly conserved macromolecular complex. Elongator consists of two subcomplexes, namely Elp123 containing the enzymatically active Elp3 subunit and the associated Elp456 hetero-hexamer. The structure of the fully assembled complex and the function of the Elp456 subcomplex have remained elusive. Here, we show the cryo-electron microscopy structure of yeast Elongator at an overall resolution of 4.3 Å. We validate the obtained structure by complementary mutational analyses in vitro and in vivo. In addition, we determined various structures of the murine Elongator complex, including the fully assembled mouse Elongator complex at 5.9 Å resolution. Our results confirm the structural conservation of Elongator and its intermediates among eukaryotes. Furthermore, we complement our analyses with the biochemical characterization of the assembled human Elongator. Our results provide the molecular basis for the assembly of Elongator and its tRNA modification activity in eukaryotes.

Cryo-EM structures of the human Elongator complex at work

“…S1 ). After 3D reconstruction and refinement, the obtained density resembles a 2-lobed architecture, known from other eukaryotic Elp123 complexes 23 , 27 . However, while one ELP123 lobe is fully resolved the second lobe is only partially visible.…”

“…While our recent work has demonstrated a functional and structural conservation of the Elongator complex from yeast ( Saccharomyces cerevisiae , Sc ) to mouse ( Mus musculus , Mm ) 23 , numerous variants of human ( Homo sapiens , Hs ) Elongator associated with pathology emphasize the need for a comprehensive characterization of the structure and tRNA modification activity of human ELP123. ELP123 can be produced in insect cells 24 , 25 , and so we first co-purified the recombinant ELP123 subcomplex with high purity, showing the expected 1:1:1 stoichiometry of the three subunits (Fig.…”

“…However, while one ELP123 lobe is fully resolved the second lobe is only partially visible. To improve the quality of the final reconstruction, we masked out the less well-defined lobe and locally refined the remaining region 23 . The final map has an overall resolution of 2.87 Å (FSC 0.143 ; Fig.…”

“…The associated Elp456 subcomplex forms a hexameric ring, which interacts asymmetrically with only one lobe of Elp123 18 , 21 , 22 . Elp456 is thought to use its ATPase activity to release the modified tRNA from the Elongator complex 18 , 23 .…”

Proteomic analysis revealed the function of PoElp3 in development, pathogenicity, and autophagy through the tRNA-mediated translation efficiency in the rice blast fungus

“…2006; Nedialkova and Leidel 2015; Tükenmez et al . 2015; Jaciuk et al . 2023), Caenorhabditis elegans (Nedialkova and Leidel 2015), and Aspergillus fumigatus (Zhang et al .…”