An open interface in the pre-80S ribosome coordinated by ribosome assembly factors Tsr1 and Dim1 enables temporal regulation of Fap7

Abstract

During their maturation, nascent 40S subunits enter a translation-like quality control cycle, where they are joined by mature 60S subunits to form 80S-like ribosomes. While these assembly intermediates are essential for maturation and quality control, how they form, and how their structure promotes quality control, remains unknown. To address these questions, we determined the structure of an 80S-like ribosome assembly intermediate to an overall resolution of 3.4 Å. The structure, validated by biochemical data, resolves a large body of previously paradoxical data and illustrates how assembly and translation factors cooperate to promote the formation of an interface that lacks many mature subunit contacts but is stabilized by the universally conserved methyltransferase Dim1. We also show how Tsr1 enables this interface by blocking the canonical binding of eIF5B to 40S subunits, while maintaining its binding to 60S. The structure also shows how this interface leads to unfolding of the platform, which allows for temporal regulation of the ATPase Fap7, thus linking 40S maturation to quality control during ribosome assembly.

The nucleoplasmic phase of pre-40S formation prior to nuclear export

“…In addition to the N terminus, in state Tsr1-2, the C-terminal domains of Tsr1 are in a flipped-out conformation, leading to the positioning of domain IV of Tsr1 at a substantial distance from h18 of the 18S rRNA and therefore unable to interact (Figure 5A ). A similar flipped-out conformation has been recently observed both in human and yeast ( 61 , 62 ), when TSR1 binds to fully matured 40S or 80S-like ribosomal complexes, respectively. Eventually, from yeast state Tsr1-3 onward, domain IV of Tsr1 interacts with immature h18 of the 18S rRNA.…”

Pseudouridine-free Ribosome Exhibits Distinct Inter-subunit Movements

“…We also purified ribosome by an affinity method in a magnesium-containing buffer that favors binding of the inactive 80S to an IgG column (Rai et al, 2021; Scaiola et al, 2018). The cells were grown similarly as described above to accumulate inactive ribosome and lysed.…”

The World of Stable Ribonucleoproteins and Its Mapping With Grad-Seq and Related Approaches

“…Alternatively, intermediates can be enriched by "starving" the assembly for one of the building blocks. This approach is widely adopted as it permits to generate higher amounts of analyzable complexes, which is particularly instrumental in studying very short-lived ribosome and spliceosome assembly stages (Li et al, 2013;Jomaa et al, 2014;Davis et al, 2016;Ni et al, 2016;Sun et al, 2017;Zeng et al, 2018;Razi et al, 2019;Seffouh et al, 2019;Du et al, 2020;Rabuck-Gibbons et al, 2020;Rai et al, 2021). However, this comes at a risk of altering the native assembly route and accumulating off-pathway, deadend particles.…”