Elongator was initially described as an RNA polymerase II-associated factor but has since been associated with a broad range of cellular activities. It has also attracted clinical attention because of its role in certain neurodegenerative diseases. Here we describe the crystal structure of the Saccharomyces cerevisiae subcomplex of Elongator proteins 4, 5 and 6 (Elp456). The subunits each show almost identical RecA folds that form a heterohexameric ring-like structure resembling hexameric RecA-like ATPases. This structural finding is supported by different complementary in vitro and in vivo approaches, including the specific binding of the hexameric Elp456 subcomplex to tRNAs in a manner regulated by ATP. Our results support a role of Elongator in tRNA modification, explain the importance of each of the Elp4, Elp5 and Elp6 subunits for complex integrity and suggest a model for the overall architecture of the holo-Elongator complex.

The eukaryotic small ribosomal subunit carries only four ribosomal (r) RNA methylated bases, all close to important functional sites. N 7 -methylguanosine (m 7 G) introduced at position 1575 on 18S rRNA by Bud23-Trm112 is at a ridge forming a steric block between P-and E-site tRNAs. Here we report atomic resolution structures of Bud23-Trm112 in the apo and S-adenosyl-L-methionine (SAM)-bound forms. Bud23 and Trm112 interact through formation of a β-zipper involving main-chain atoms, burying an important hydrophobic surface and stabilizing the complex. The structures revealed that the coactivator Trm112 undergoes an induced fit to accommodate its methyltransferase (MTase) partner. We report important structural similarity between the active sites of Bud23 and Coffea canephora xanthosine MTase, leading us to propose and validate experimentally a model for G1575 coordination. We identify Bud23 residues important for Bud23-Trm112 complex formation and recruitment to pre-ribosomes. We report that though Bud23-Trm112 binds precursor ribosomes at an early nucleolar stage, m 7 G methylation occurs at a late step of small subunit biogenesis, implying specifically delayed catalytic activation. Finally, we show that Bud23-Trm112 interacts directly with the box C/D snoRNA U3-associated DEAH RNA helicase Dhr1 supposedly involved in central pseudoknot formation; this suggests that Bud23-Trm112 might also contribute to controlling formation of this irreversible and dramatic structural reorganization essential to overall folding of small subunit rRNA. Our study contributes important new elements to our understanding of key molecular aspects of human ribosomopathy syndromes associated with WBSCR22 (human Bud23) malfunction.ribosome synthesis | rRNA modifying enzyme | methyltransferase | translation | S-adenosyl-L-methionine

Most of the factors involved in translation (tRNA, rRNA and proteins) are subject to post-transcriptional and post-translational modifications, which participate in the fine-tuning and tight control of ribosome and protein synthesis processes. In eukaryotes, Trm112 acts as an obligate activating platform for at least four methyltransferases (MTase) involved in the modification of 18S rRNA (Bud23), tRNA (Trm9 and Trm11) and translation termination factor eRF1 (Mtq2). Trm112 is then at a nexus between ribosome synthesis and function. Here, we present a structure-function analysis of the Trm9-Trm112 complex, which is involved in the 5-methoxycarbonylmethyluridine (mcm5U) modification of the tRNA anticodon wobble position and hence promotes translational fidelity. We also compare the known crystal structures of various Trm112-MTase complexes, highlighting the structural plasticity allowing Trm112 to interact through a very similar mode with its MTase partners, although those share less than 20% sequence identity.