The Rps15 carboxy-terminal tail helps recruit Tsr3. (A) Predicted binding site of Tsr3 on 80S-like ribosomes. Pre-40S (PDBID: 6ZXE) and 80S-like (PDBID: 6WDR) ribosome structures were first overlayed based on 18S rRNA. The crystal structure of Tsr3 (PDBID: 5APG) was aligned to Rio1 from pre-40S (PDBID: 6ZXE) and then rotated so that its positively charged surface and its substrate-binding pocket face its rRNA substrate, U1191. 18S rRNA from 80S-like ribosome (PDBID: 6WDR) is shown in gray ribbon, with U1191 highlighted in green sphere. Rps15 from 80S-like ribosome is shown in yellow, with residues Y123, R127, and R130 highlighted in sphere. Tsr3 is shown as surface and colored based on charge (blue: positive; red: negative). The active site of Tsr3 is indicated by a black arrow based on its bound substrate analog, Se-adenosyl-selenomethionine. (B) (Top) Primer extension results show acp modification at U1191 of ribosomes from Gal:S15 cells supplemented with plasmids encoding WT S15 or S15_YRR and empty vector (EV) or Tsr3. (Bottom) Quantifications of the primer extension stop at U1191 relative to the full extension from the top and additional replicates. Significance was tested using one-way ANOVA (Sidak's multiple comparisons test). (****) Padj < 0.0001; n ≥ 6. (C) (Top) Primer extension results show acp modification at U1191 of ribosomes from Gal:Rps3 cells supplemented with plasmids encoding WT Rps3 or Rps3_KK and empty vector (EV) or Tsr3. (Bottom) Quantifications of the primer extension stop at U1191 relative to the full extension from the top and additional replicates. Significance was tested using one-way ANOVA (Sidak's multiple comparisons test). (*) Padj < 0.05; n = 3.
