Purification of Hibernating and Active C− Ribosomes from Zinc-Starved Mycobacteria

Starvation sensing by mycobacterial RelA/SpoT homologue through constitutive surveillance of translation

“…The crude ribosome preparations were then layered on top of 37 mL sucrose density gradients (10%-40%) and centrifuged for 16 hours at 24,000 rpm in a Beckman rotor SW 28. The 70S ribosome fractions were collected after fractionating the sucrose gradient in a 260 nm Brandel gradient analyzer as previously described (Li et al, 2021a). The pooled 70S ribosome fractions pelleted by ultracentrifugation at 42,800 rpm for 3 hours in a Beckman rotor Type 70Ti were suspended in HMA-10 buffer (20 mM HEPES-K pH7.5, 30 mM NH 4 Cl, 10 mM MgCl 2 , 5 mM β-mercaptoethanol) and quantified by measuring absorbance at 260 nm.…”

“…The reaction was then incubated for an additional 30 minutes at 37°C to allow joining of the 50S subunit. Resulting complexes were either used for structural analysis or resolved on a 15 mL 10-40% sucrose density gradient centrifuged at 35000 rpm for 135 minutes using SW41 rotor on an Optima L-90K ultracentrifuge (Beckman Coulter), and the resolved particles were fractionated as previously described (Li et al ., 2021a). For immunoblotting analysis, content of the indicated fractions were precipitated using Methanol-chloroform extraction as previously described (Li et al ., 2020).…”