Supplementary Figure 2: Monitoring of the integrity of RNCs.
From: A structural ensemble of a ribosome–nascent chain complex during cotranslational protein folding
An integrated approach using NMR and biochemical analyses was used to determine the integrity of the RNCs over time. (a) For each of FLN5+21, 45, 47, 67 and 110 RNCs, the 1H-13C correlation spectrum is shown (identical to Fig. 3a), overlaid with that of isolated FLN5 (cyan). Resonances arising from labelled 70S proteins can be observed in spectra at ca. δH=0.8 p.p.m. (b) The integrity of these RNCs was monitored over time and the timeframe during which the nascent chain was determined to be attached in each RNC as derived by NMR methods is indicated by the shaded region: 13C-edited diffusion experiments (1H STE-1H,13C-HMQC) were used to determine the diffusion coefficient associated with the nascent chain in FLN5+67 and 110 RNCs (cyan). The changes in intensities of the cross-peaks observed in the correlation spectra were used to monitor the FLN5+45 and 47 RNCs (green) and the diffusion coefficients (from 13C-edited experiments) of the combined ribosome-derived resonances were monitored for FLN5+21 RNC (yellow). (c) Western blots against either the N-terminal His-tag or C-terminal SecM sequence report on both the tRNA-bound and released forms of the nascent chain. (d) Densitometry analysis of the tRNA-bound form over time as assessed by the anti-His western blot. In the analyses shown in panels b, c and d, the shaded region represents the timeframe corresponding to an exclusively ribosome-bound nascent chain and for which the 2D correlation spectra are summed and presented in panel a. (e) As a representative example for 15N-labelled RNCs, a 1H-15N SOFAST-HMQC spectrum (identical to that in Fig. 3b) of FLN5+21 RNC (black) is overlaid with a 2D correlation spectrum of isolated FLN5∆12 (blue) and a selection of unambiguously assigned resonances is indicated. (f) Diffusion coefficient for FLN5+21 RNC resonances assessed over time (calculated from 15N XSTE spectra; single spectrum shown on left). A signal attenuation (I95/I5) by a factor of > 0.62 corresponds to a diffusion coefficient of an intact ribosomal particle (D = 2.0 ± 0.3 10-11 m2 s-1) at 25°C. The timeframe during which the nascent chain is assessed as being intact is shaded in grey. (g) 15N XSTE spectra of FLN5+21 RNC recorded at gradient strengths 5% and 95% of the maximum gradient strength Gmax, 0.557 T m-1 for the timeframe during which the nascent chain was deemed to be intact. (h) Anti-His and anti-SecM western blot analyses of FLN5+21 RNC.
