Loss of SRL inhibits assembly of 50S subunits in vivo but not in vitro. (A) Sucrose gradient traces of reconstituted control subunits (blue), reconstituted ΔSRL subunits (orange), and control 50S subunits isolated from cells (black). The term “control” is used here to specify the aptamer-tagged wild-type case. (B) PT activity of isolated and reconstituted 50S particles, containing or lacking the SRL (as indicated). Purified 30S subunits were used as a negative control (gray, as indicated). (C) Gain of PT activity during step 2 of the reconstitution protocol, for control and ΔSRL cases (as indicated). Shown are data from four independent experiments, and corresponding best-fit exponential curves. Observed rates (control, 0.022 ± 0.006 min−1; ΔSRL, 0.012 ± 0.006 min−1) are statistically indistinguishable. (D) PT activity of control and ΔSRL particles isolated from cells before and after incubation at 50°C in the presence or absence of excess TP50 (as indicated). Bars represent the mean ± SEM of three independent experiments, and individual data points (circles) are superimposed. Asterisks indicate P < 0.05, based on an unpaired two-tailed t-test. (ns) Not significant.
