Polysome profiling

To perform polysome profiling, 200 ml of L. lactis IL1403 Int⁺/Int^- cells (1:20 dilution of saturated overnight culture) were grown and induced for gene expression with nisin for 2–3 hr. 100 mg/ml chloramphenicol was added and the cultures were chilled on ice for ~30 min with intermittent whirling and then collected by centrifugation at 5,000 rpm for 10 min at 4°C. Cell pellets were resuspended in 500 μl of ice-cold lysis buffer (20 mM Tris-HCl, pH 8.0; 140 mM KCl; 40 mM MgCl₂; 0.5 mM DTT; 100 μg/ml chloramphenicol; 1 mg/ml heparin; 20 mM EGTA; 1% Triton X-100) and washed twice with the same buffer. The cell pellets were resuspended again in 500 μl of lysis buffer and snap-frozen in liquid nitrogen. Then the cells were disrupted at 4°C in 15 ml falcon tubes with 500 μl of 0.1 mm ice-cold glass beads by rigorous vortexing (30 s for 20 times, with 1 min interval) and then briefly spun down at 4,000 rpm for 5 min. Crude cell lysate was then gently mixed, transferred into 1.5 ml tubes on ice, and cleared by 14,000 rpm for 25 min at 4°C. 400 μl of the cleared lysates were then loaded onto prepared 10–50% sucrose gradients in lysis buffer without 0.5 mg/ml heparin and centrifuged at 36,000 rpm for 153 min at 4°C (SW41 rotor). 30 fractions of ~400 μl each were collected for each gradient from top to bottom. RNAs were extracted from each fraction by using phenol/chloroform and analyzed by running gels and performing Northern blotting.