Fig. 3. Ribosome-bound Get4 contacts h46/47 of the 25S rRNA.

a Crosslinking and analysis of cDNA (CRAC). (I) Yeast expressing His₆-TEV-ProtA-tagged (HTP-tagged) Get4 (Get4-HTP) was grown in the presence of 4-thiouracil (4sU) and was then crosslinked via irradiation at 365 nm. (II) Get4-HTP-containing complexes were purified under native conditions, RNA was trimmed, and TEV protease-mediated cleavage was performed. (III) RNA fragments were [³²P]-labeled, adaptors were ligated, His₆Get4-containing crosslink products were denatured, and purified via Ni-NTA. (IV) cDNA libraries were generated, (V) amplified by PCR, and sequenced. Yeast cell (pink), ribosome (gray), polypeptide tunnel exit (black), crosslinked rRNA (red), Get4/5 as in Fig. 2a. b Get4 contacts RNA. ProtA-purified, trimmed, [³²P]-labeled, and crosslinked material (ProtA immunoblot, upper panel) and 6 h autoradiography ([³²P]-labeled RNA, lower panel) of the indicated strains. Membrane regions, which were employed for further analysis as shown in panels c and d are boxed in red. c Get4 contacts 25S rRNA h46/h47. Sequencing reads mapping to nucleotides of the S. cerevisiae RDN37 rDNA sequence. Get4-HTP (red) and wild type (black). Shown is a representative experiment of two biological replicates (accession code GSE151664). PCR amplification (panel a, step V) was 35 cycles for wild type, and 24 cycles for Get4-HTP. d Heat map of Get4 contacts to 25S rRNA. 25S rRNA secondary structure and zoom into the rRNA region crosslinked to Get4⁷². Red (100%) indicates the maximum number of reads obtained, lower numbers of reads (>20%) are shown in shades of orange–yellow. e Contacts between Get4/5, ribosomal proteins, and rRNA. Surface representation of the yeast 80S ribosome (PDB 4V88⁷⁸). 60S (light gray) and 40S (gray). Contacts of Get5 to ribosomal proteins close to the tunnel exit: Rpl26 (deep purple), Rpl35 (dirty violet), Rpl37 (hot pink). A red circle indicates the ribosomal tunnel exit. Additional contacts of Get5 are shown in Supplementary Fig. 3j and Table 1. f Zoom into the ribosomal tunnel. Color code as in panel e. The tunnel exposed β-sheet of Rpl17 is indicated in black. Contacts of Get4 to h46/h47 of the 25S rRNA are as in d (see Supplementary Fig. 3k).