posted on 2022-01-18, 17:35authored byUdit Dalwadi, Dhiraj Mannar, Felix Zierhut, Calvin K. Yip
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.