Bacterial culture conditions and lysis

E. coli MG1655 cells were grown overnight at 37°C in MOPS EZ Rich Defined media (Teknova) supplemented with 0.2% glucose and diluted 1:100 into 300 mL fresh media and grown at 37°C to an optical density of 0.3. Cultures were treated with 200 µM mupirocin (MPC, Sigma) or 1 mM chloramphenicol (Cm, Sigma) when indicated in the text. Cells were harvested either by filtration or by direct freezing of the culture in liquid nitrogen. Biological replicates consist of cultures from individual colonies grown on separate days.

Filtration was performed using a Kontes 90 mm filtration apparatus with 0.45 µm nitrocellulose filters (Whatman); cells were scraped from the filter before the media runs dry and were then frozen in liquid nitrogen. 0.65 mL of frozen lysis buffer was added to the pellets as indicated in the text. The standard lysis buffer is 20 mM Tris pH 8.0, 10 mM MgCl₂, 100 mM NH₄Cl, 5 mM CaCl₂, 0.1% NP‐40, 0.4% TritonX‐100, and 100 U/mL DNase I (Roche). To this buffer, 1 mM chloramphenicol, 1 M NaCl or 150 mM MgCl₂ was added as indicated in the text. The cells were cryogenically pulverized using a Spex 6870 freezer mill with 5 cycles of 1 min grinding at 5 Hz and 1 min cooling. Lysates were thawed at room temperature and gently homogenized by passing through a 20 gauge syringe five times. Lysates were clarified by centrifugation at 20,000 g for 10 min at 4°C. For buffer exchange, 25 AU of RNA in the lysates was layered on top of a 1 mL sucrose cushion (20 mM Tris pH 7.5, 500 mM NH₄Cl, 0.5 mM EDTA, 1.1 M sucrose) and ribosomes were pelleted by centrifugation using a TLA 100.3 rotor at 65,000 rpm for 2 hr. Pellets containing ribosomes were re-suspended using resuspension buffer (0.2 mL of 20 mM Tris pH 8.0, 10 mM MgCl₂, 100 mM NH₄Cl, 5 mM CaCl₂, 0.1% NP‐40, 0.4% TritonX‐100) and used for subsequent experiments.

For samples harvested by direct freezing, 50 mL of culture at OD₆₀₀ of 0.3 was directly sprayed from a pipette into liquid nitrogen. The frozen culture was cryogenically pulverized together with 5.6 mL 10x lysis buffer (1x concentrations listed above) with 10 cycles of 1 min grinding at 8 Hz and 1 min cooling. Lysates were thawed at room temperature and pelleted over a 3 mL sucrose cushion (1.1 M sucrose, 20 mM Tris pH 8, 500 mM NH₄Cl, 10 mM MgCl₂, 0.5 mM EDTA) using a Ti-70 rotor at 70,000 rpm for 2 hr. Ribosome pellets were re-suspended in 200 µL resuspension buffer.