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Elucidating the structural and chemical mechanisms of tRNA carboxymethylation by the radical SAM enzyme ELP3
Date
2025
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Publisher
University of Delaware
Abstract
Transfer RNAs (tRNAs) are critical intermediaries in protein translation, ensuring translational efficiency and fidelity, and feature a variety of posttranscriptional chemical modifications that fine tune their functions. A key group of these modifications are the 5-carboxymethyluridine (cm5U34)-derived modifications found at wobble uridines in the anticodons of several different tRNAs. These modifications are crucial for maintaining protein translation and cellular health and are linked to a wide variety of diseases and disorders, including various cancers and neurodegenerative diseases. In eukaryotes, the cm5U parent modification is installed by the multiprotein Elongator complex, whose catalytic subunit Elp3 is conserved across all domains of life. Elp3 is composed of a [4Fe-4S] cluster-bearing radical SAM domain (rSAM) and a lysine acetyltransferase (KAT) domain, which together install cm5U on tRNA using radical-based chemistry and the cofactors S-adenosylmethionine (SAM) and acetyl-Coenzyme A (AcCoA). Despite extensive research, the molecular details of how Elp3 catalyzes cm5U formation have remained unclear. Previous mechanistic work has proposed multiple potential chemical mechanisms for cm5U formation, with available data unable to distinguish between them. Separately, structural work has revealed a 20+ Å distance between the rSAM and KAT active sites, with minimal research focused on how Elp3 bridges this distance to install cm5U on tRNA. Here I reconstitute in vitro Elp3-mediated tRNA modification and present a combination of structural analyses with mutational and isotopic labeling assays that provide new insight into Elp3’s mechanism. My results reveal that Elp3 harbors a conserved molecular tunnel that it uses to shuttle acetate between its two distant active sites, suggesting a new model for Elp3’s molecular mechanism.
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Keywords
Transfer RNAs, Acetyl-Coenzyme A, S-adenosylmethionine, Molecular mechanism