Figure 2. Protein-ribosome interaction on a microwell.

eIF6 off-rate is different in pre-ribosomes and mature ribosomes.(a) Experimental design with immobilized 60S subunits and labeled eIF6. (b) Saturation curve of eIF6 binding to 60S. Binding performed at room temperature. (c) 50% displacement by ten-fold unlabeled eIF6 coincubated with labeled eIF6. (d) Dependence of eIF6 binding to 60S by Mg²⁺. (e) On-rate. (f) Off-rate of eIF6 from mature ribosomes, shows eIF6 release in the first 30 min. All the experiments b-f were performed at least five times. Mean + SD. (g) Scheme of ribosomal maturation with eIF6/Tif6 bound both to pre-ribosomes in the nucleus and mature ribosomes in the cytoplasm. eIF6 is added to preribosomes in the nucleolar/nucleus compartment and exported to the cytoplasm. In the cytoplasm 60S subunits are inactive until eIF6 is released. (h) Stripping of endogenous eIF6 from purified adsorbed pre-ribosomes leads to increased exogenous eIF6 binding to pre-ribosomes. eIF6 binding to nucleolar ribosomes before (black) and after (grey) stripping of endogenous eIF6 with 300 mM KCl. (i) eIF6 off-rate from pre-ribosomes, loaded with exogeneous eIF6 after stripping of the endogenous one. All the experiments were performed at least five times. Representative curves are shown. Mean + SD. T-Test, paired, two-tailed.