Supplementary Figure 1: Design of isolated protein and RNC constructs, and homogeneity of purified RNCs.

(a) Schematic depicting the design and nomenclature used for all the isolated proteins and RNCs used in this study. Sequence boundaries are indicated. The isolated FLN constructs comprise: a range of FLN5 C-terminal truncations in which between 2 and 21 amino acids have been removed, “FLNΔX”, in which X refers to the extent of the truncation, the folding incompetent variant “FLN5 Y719E” harboring a destabilizing Glu mutation at the site indicated in orange, and “FLN5-6Δ18” in which FLN6 has the removal of 18 amino acids from its C-terminus (Hsu, S.T. et al. Proc Natl Acad Sci U S A 104, 16516-21, 2007). In SecM-stalled FLN5 RNCs, the FLN5 sequence is tethered to the PTC via increasing lengths of the FLN6 sequence and the SecM translational arrest motif; for simplicity this will be referred to here as the “linker”. The linker, ranging from 21 to 110 amino acids, therefore corresponds to the distance (in residues) between its most C-terminal residue, G750, and the PTC. Folding-incompetent RNCs have the Y719E mutation in FLN5 and are referred to as “FLN5+L Y719E RNCs”. For the poly(GS) RNCs, FLN6 is replaced with a repeating poly(GS) motif, GGGG/S, to generate equivalent linkers of between 21 to 67 residues in length, as present in the FLN5 RNCs. (b) Representative PAGE gel of a purified RNC (FLN5+42) and of 70S ribosomes visualized with Coomassie blue stain (left) and silver stain (right). The gels show a banding pattern characteristic of ribosomal proteins and show that the RNCs are essentially free of extraneous proteins. The bands corresponding to ribosomal proteins L1, L2, L6, S5, L29, L31, L35 are highlighted. (c) An anti-His western blot of FLN5+42 RNC and of 70S ribosomes, showing the tRNA-bound form of the RNC and the absence of any non-specific detection of ribosomal proteins in untranslating 70S ribosomes. The extent of nascent chain attachment to ribosomes was determined to be > 90% across all samples. (d) The amount of residual trigger factor (TF) and DnaK present within the purified RNC samples was assessed by densitometry analysis of anti-TF and anti-DnaK western blots, respectively. The purified samples are essentially free of the effects of TF and DnaK (≤ 1.2%). Highlighted in asterisks are the RNCs for which TF or DnaK levels were below levels of detection. (e) Representative western blots of RNC samples which were presented in a modified form in Fig. 2c are shown here for clarity, where the RNCs shown in Fig. 2c are marked by asterisks.