Conserved from yeast to humans and composed of six core subunits (Elp1-Elp6), Elongator is a multiprotein complex that catalyzes the modification of the anticodon loop of transfer RNAs (tRNAs) and in turn regulates messenger RNA decoding efficiency. Previous studies showed that yeast Elongator consists of two subassemblies (yElp1/2/3 and yElp4/5/6) and adopts an asymmetric overall architecture. Yet, much less is known about the structural properties of the orthologous human Elongator. Furthermore, the order in which the different Elongator subunits come together to form the full assembly as well as how they coordinate with one another to catalyze tRNA modification is not fully understood. Here, we purified recombinant human Elongator subunits and subassemblies and examined them by single-particle electron microscopy. We found that the human Elongator complex is assembled from two subcomplexes that share similar overall morphologies as their yeast counterparts. Complementary co-purification and pulldown assays revealed that the scaffolding subunit human ELP1 (hELP1) has stabilizing effects on the human ELP3 catalytic subunit. Furthermore, the peripheral hELP2 subunit appears to enhance the integrity and substrate-binding ability of the dimeric hELP1/2/3. Lastly, we found that hELP4/5/6 is recruited to hELP1/2/3 via hELP3. Collectively, our work generated insights into the assembly process of core human Elongator and the coordination of different subunits within this complex.
© 2022 The Authors. Published by American Chemical Society.