Fig. 5

Mak5 crosslinks to the 25S rRNA sequence that is later bound by Rpl10. a The number of sequence reads in the Mak5-HTP CRAC data corresponding to each nucleotide of the 25S rRNA sequence is shown on the secondary structure of the mature 25S rRNA using a colour code where the maximum number of reads (100%) is shown in red and lower numbers of reads (10%) are shown in yellow. A magnified view of the Mak5 crosslinking sites in H36–37 and H39 is shown. b The number of sequence reads in the Mak5-HTP CRAC data corresponding to each nucleotide of the 25S rRNA sequence was modelled onto the tertiary structure of the 25S rRNA sequence in a pre-60S complex purified via Nsa1 and Nop2 (state 3; PDB: 6CB1 [https://www.rcsb.org/structure/6CB1]) using a colour code as in (a). Densities corresponding to specific ribosomal proteins and pre-60S biogenesis factors are highlighted in various colours. c Nop2-containing pre-60S complexes purified from yeast expressing (+) or depleted (−) of Mak5 were immobilised on IgG sepharose and either treated with DMS (+) or left untreated (−). RNAs isolated from these complexes served as substrate for primer extension reactions and cDNA fragments were separated by denaturing PAGE, alongside a sequencing ladder, before visualisation. Differentially methylated nucleotide are indicated. Replicate experiments were performed and representative data is presented. d Mature 25S rRNA and pre-60S complexes isolated via different TAP or HTP-tagged biogenesis factors that bind sequentially to early, intermediate and late pre-ribosomal complexes as indicated were immobilised on IgG sepharose and then either treated with DMS (+) or left untreated (−). RNAs isolated from these complexes were used for primer extension reactions, and cDNA fragments were separated by denaturing PAGE and visualised using a phosphorimager. e The number of sequence reads in the Mak5-HTP CRAC data corresponding to each nucleotide of the 25S rRNA sequence was modelled onto the tertiary structure of the mature 60S subunit (PDB: 4V88 [https://www.rcsb.org/structure/4V88]) using a colour code as in (a). The ribosomal protein Rpl10 is highlighted in blue and a magnified view of the contacts between 25S-H39 and Rpl10 is shown