Molecular basis of tRNA recognition by the Elongator complex

Abstract

The highly conserved Elongator complex modifies transfer RNAs (tRNAs) in their wobble base position, thereby regulating protein synthesis and ensuring proteome stability. The precise mechanisms of tRNA recognition and its modification reaction remain elusive. Here, we show cryo-electron microscopy structures of the catalytic subcomplex of Elongator and its tRNA-bound state at resolutions of 3.3 and 4.4 Å. The structures resolve details of the catalytic site, including the substrate tRNA, the iron-sulfur cluster, and a SAM molecule, which are all validated by mutational analyses in vitro and in vivo. tRNA binding induces conformational rearrangements, which precisely position the targeted anticodon base in the active site. Our results provide the molecular basis for substrate recognition of Elongator, essential to understand its cellular function and role in neurodegenerative diseases and cancer.

Fig. 1. Cryo-EM structure of yeast Elp123 showing its active site.

(A) Atomic model of Elp123 shown from front and top. The FeS cluster (orange and yellow) and the 5′dA molecule (green) are highlighted. Elp1, Elp2, and Elp3 domain organization is shown schematically and labeled in the upper panel. Scale bar, 25 Å. (B) Close-up of the active site of Elp3. Model and respective density of the [4Fe4S] cluster and the 5′dA molecule are shown. The coordinating cysteine residues are labeled, and the additional density next to the fourth iron atom is indicated by a question mark (upper left). Residues involved in FeS cluster and SAM coordination are shown with their respective densities (lower left). Organization of the active site including ligands and involved protein residues (right).

Fig. 2. Cryo-EM structure of Elp123 bound to tRNA^Ala_UGC.

(A) EMSA assay using endogenous Elp123 (left), recombinant Elongator (right), and fluorescently labeled tRNA^Ala_UGC in the presence of different concentrations of glutaraldehyde. The positions of free tRNA and protein-tRNA complexes (*) are indicated next to the native polyacrylamide gel electrophoresis (PAGE). (B) Representative microscale thermophoresis measurements, respective fits, and calculated dissociation constant (K_d) values for Elp123 (blue) and Elongator (green). In both cases, the Hill coefficient is close to 1, indicating the presence of independent binding sites. n = 3. (C) Atomic model of Elp123 lobe bound to tRNA (Elp1, orange; Elp2, yellow; Elp3, violet) showing additional electron density for tRNA at a resolution of 4.4 Å. The fitted tRNA^Ala_UGC model is colored in accordance to the scheme on the right [purple, acceptor stem (Acc); red, D-loop (DL); blue, ASL; orange, variable loop (VL); green, T-loop (TL)]. Close-up view highlighting the phosphate backbone of the VL and ASL following the density (bottom right). (D) Atomic model of Elp123-tRNA from different perspectives.

Fig. 3. Elp123 compaction upon tRNA binding.

(A) Superimposition of Elp123 (gray) and Elp123-tRNA (orange/light orange, yellow, violet) structures. Observed differences are indicated by arrows. (B) Model (left) and density (right) of Elp123 bound to two tRNA molecules at the same time. Subunits and tRNA molecules (blue) are labeled.

Fig. 4. Structural details of tRNA binding to Elp123.

(A) Structural superimposition of tRNA-bound (violet) and unbound Elp3 (gray). The contact points of neighboring Elp1 and Elp2 subunits are shown. r.m.s.d., Root-mean-square deviation. (B) Superimposition and close-up of the active site in the unbound (gray) and tRNA-bound (colored) Elp123 structure, highlighting potentially shifted residues (in bold). (C) Structural comparison between the Elp123-bound tRNA^Ala_UGC (middle) and different structural models from different templates (tRNA^Phe_GAA PDB ID: 1EHZ, left; tRNA^Glu_UUC PDB ID: 5HR6, right). Below, the differences between the ASL region of tRNA^Phe_GAA (left) and tRNA^Ala_UGC bound to Elp123 are highlighted. For the Elp123-tRNA structure, the density in the ASL region is shown and the individual bases are labeled. (D) Close-up view of the ASL region bound to the central cavity of Elp3. Conserved basic residues and RNA bases in the anticodon loop are labeled.

Fig. 5. Functional validation of Elp123.

(A) Mean acetyl-CoA (ACO) hydrolysis rate of endogenous ScElp123 (blue) and recombinant Elongator (green) in the presence of different tRNAs. Statistical significance was determined by ordinary two-way analysis of variance (ANOVA; α = 0.05) followed by Tukey’s honestly significant difference test applied to the entire dataset. ns, not significantly different from the protein samples without tRNA (horizontal dotted line); **P < 0.01; mean ± SEM; n = 3; in the case of elp3Δ bulk tRNA, n = 6. (B) Structural overview of all mutations, truncations, and deletions tested in yeast. (C) Phenotypic analyses of various yeast strains carrying variations in Elp1, Elp2, and Elp3 using zymocin resistance assays.

Fig. 6. Reaction and regulatory mechanisms of Elongator.

(A) Overview of intermediate steps carried out by Elp3 during the cm⁵ tRNA modification reaction cycle. The [4Fe4S] cluster (orange/yellow; FeS) bound in the SAM domain of Elp3 and the acetyl-CoA (ACO, green) molecule bound in the KAT domain of Elp3 are labeled. (B) Schematic illustration of Elp123 structural rearrangements upon tRNA binding and Elp456 interaction. (C) Model of the Elp123-tRNA and Elongator lobe bound to tRNA fitted into the negative-stain densities of Elp123 and Elongator. (D) Tilted view of tRNA-bound Elongator lobe model highlighting the two main contact points. The possibility of adenosine triphosphate (ATP)–mediated tRNA release is indicated. ADP, adenosine diphosphate.