The Ribosome Assembly Factor Reh1 is Released from the Polypeptide Exit Tunnel in the Pioneer Round of Translation

Musalgaonkar S. et al.

Assembly of functional ribosomal subunits and successfully delivering them to the translating pool is a prerequisite for protein synthesis and cell growth. In S. cerevisiae, the ribosome assembly factor Reh1 binds to pre-60S subunits at a late stage during their cytoplasmic maturation. Previous work shows that the C-terminus of Reh1 inserts into the polypeptide exit tunnel (PET) of the pre-60S subunit. Unlike canonical assembly factors, which associate exclusively with pre-60S subunits, we observed that Reh1 sediments with polysomes in addition to free 60S subunits. We therefore investigated the intriguing possibility that Reh1 remains associated with 60S subunits after the release of the anti-association factor Tif6 and after subunit joining. Here, we show that Reh1-bound nascent 60S subunits associate with 40S subunits to form actively translating ribosomes. Using selective ribosome profiling, we found that Reh1-bound ribosomes populate open reading frames near start codons. Reh1-bound ribosomes are also strongly enriched for initiator tRNA, indicating they are associated with early elongation events. Using single particle cryo-electron microscopy to image cycloheximide-arrested Reh1-bound 80S ribosomes, we found that Reh1-bound 80S contain A site peptidyl tRNA, P site tRNA and eIF5A indicating that Reh1 does not dissociate from 60S until early stages of translation elongation. We propose that Reh1 is displaced by the elongating peptide chain. These results identify Reh1 as the last assembly factor released from the nascent 60S subunit during its pioneer round of translation.

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October, 2023


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