The conserved β‑hairpin of the SUI1 domain is a dual-function structural module governing translation initiation and ribosome recycling in yeast

<jats:p>Most eukaryotic mRNAs encode a single functional polypeptide. Following translation termination, both the large and small ribosomal subunits are typically released from the mRNA by ribosome recycling factors. However, after translating short upstream open reading frames (uORFs) within the 5' untranslated regions (UTRs), ribosomes can remain associated with the mRNA and reinitiate translation. This process is regulated by the heterodimer MCTS1·DENR (Tma20p·Tma22p in yeast). DENR/Tma22p harbors a SUI1 domain, structurally homologous to the translation initiation factor eIF1/Sui1p, which features a conserved, positively charged β-hairpin loop critical for eIF1 function. Despite this structural similarity, the functional significance of specific elements within DENR/Tma22p remains unexplored. Here, we used in vivo reporter assays in Saccharomyces cerevisiae to quantify reinitiation efficiency following translation of either a short uORF (in the 5' UTR) or a full-length coding sequence (in the 3' UTR). Systematic analysis of single, double, and triple deletions of TMA20, TMA22, and TMA64 (a homolog of Tma20p·Tma22p) revealed that the Tma20p·Tma22p complex exerts a dominant role over Tma64p in modulating reinitiation, while exhibiting functional interplay between the two factors. Using knockout strains complemented with Tma22p variants, we further demonstrated that the positively charged residues of the β-hairpin loop 1 are essential for Tma22p recycling activity. Unexpectedly, deletion of the entire SUI1 domain was less deleterious, and eIF1/Sui1p was able to partially substitute for the SUI1 domain of Tma22p within a chimeric protein context. Our findings establish the β-hairpin loop 1 of the DENR/Tma22p SUI1 domain as a critical determinant for ribosome recycling and reinitiation, and raise the question of whether MCTS1/Tma20p can promiscuously operate with both DENR/Tma22p and eIF1/Sui1p - two specialized factors that evolved from a common structural scaffold to govern distinct steps in the translation cycle.</jats:p>