Fig. 8: Coordination of GroEL with Trigger factor and DnaK.

a Multi-chaperone co-sedimentation assay. RNCs were incubated with GroEL and either TF or DnaK/DnaJ/ATP before centrifugation through a 35% sucrose cushion to separate the ribosomal fraction (pellet) from unbound chaperones (supernatant). b TF outcompetes GroEL at short NCs. Coomassie-stained SDS-PAGE of the resuspended ribosomal pellet from the co-sedimentation assay. RNC_1-180 or RNC_333-510 were incubated with GroEL (+EL), Trigger factor (+TF), or both chaperones together (+EL + TF). Bands corresponding to the NC (*), TF, GroEL, and ribosomal proteins (r.p.) are indicated. The experiment was repeated twice with similar results. c TF and GroEL do not compete for binding long NCs. Chaperone co-sedimentation with RNC_1-1014 was tested as in (b). The experiment was repeated twice with similar results. d GroEL and DnaK compete for binding RNCs. Coomassie-stained SDS-PAGE of the resuspended ribosomal pellet from the co-sedimentation assay as in (a). RNC_1-510WT or RNC_1-510mut were incubated either with GroEL (+EL), DnaK/DnaJ/ATP ( + KJ) or both (+EL + KJ). Bands corresponding to the NC (*), DnaK, GroEL, and ribosomal proteins (r.p.) are indicated. The experiment was repeated twice with similar results. e Model for the cotranslational action of GroEL/ES. Poorly folded NCs (i) are captured by the apical domains and C-terminal tails of GroEL or EL:ES₁, resulting in further unfolding (ii and iii). GroEL may dissociate, or the cis ring may be capped by GroES to partially encapsulate the ribosome-bound NC (iv). In this state it is possible that the trans ring may capture a second NC or post-translational client. The NC may then fold enough to exclude GroEL/ES, or remain encapsulated and complete folding in the chaperonin cavity post-translation (v). Some substrates may only become encapsulated after translation termination (arrow from iii to v). Source data are provided as a Source Data file.