Fig. 7: Action of ribosome-associated Tig2.

a Left: Schematic workflow of ribosome co-sedimentation assay. Immunoblot of total protein (T) as input control, supernatant fraction (S), and the pellet sample (R) containing the enriched ribosomes. Samples derive from 3-week-old densely pot-grown plants, treated with standard ribosome buffer (Mg²⁺), with 800 mM KCl (high salt) or 1 mM puromycin (Puro). Right: Quantification of immunoblot (R/(R + S)) from three independent experiments, mean values, SD and two-sided Student’s t-test p-values are given. b Structural prediction of the Arabidopsis 23S rRNA, relevant ribosomal protein surrounding the tunnel exit site and Tig2, based on AlphaFold 3. Predicted Arabidopsis models are superimposed with the Cryo-EM 6ERI structure of spinach³⁴. Postulated Tig2-ribosome interaction is shown in the right panels with Tig2 shown as surface charge (top) and ribbon model (bottom). Predicted contacts between amino acids of Tig2 and uL24c are shown. c and d Mass spectrometric quantification of proteins that were present in ribosomal pellets of the category chloroplast ribosome biogenesis (b) and co-translational protein maturation (c). Col-0 and tig2 lines were kept for 21 days in the cold (4 °C) or at standard conditions (21 °C) for two weeks. Heatmaps represent fold-change differences (log₂) between Col-0 and tig2 lines. e Left: model of the chloroplast ribosome derived from the 6ERI Cryo-EM structure³⁴. Relevant ribosomal proteins surrounding the tunnel exit site are marked in color; positions of 23S rRNA breaks are indicated in red. RPS ribosomal protein of the small subunit, RPL ribosomal protein of the large subunit. Right: Schematic overview of final rRNA maturation and the role of RH39 according to²⁹. Postulated action of Tig2 is indicated.