Figure 2.

Dissociation of mRNAs from ribosomes upon stress. Cell lysates were prepared as described in Materials and Methods, loaded on a sucrose gradient (7–50%) in a SW41 ultracentrifuge tube. Contents were separated by centrifugation at 35 krpm for 3 h at 4°C and fractionated by isotonic pumping with online reading of A₂₈₀. The bottom of the gradients, representing high-molecular-weight fractions, is oriented to the right. (A) Response to tBOOH or NaCl in rck2Δ cells. Cultures were harvested after growth for 30 min in the presence of 0.8 mM tBOOH or for 15 min after addition of NaCl to 0.4 M. Solid black line, wild-type cells, no stress; dashed black line, wild-type cells with tBOOH; dotted black line, wild-type cells after 0.4 M NaCl; solid gray line, rck2Δ cells, no stress; dashed gray line, rck2Δ cells with tBOOH; and dotted gray line, rck2Δ cells after 0.4 M NaCl. (B) Time course after exposure to tBOOH in cells expressing rck2-kd. Cells were exposed to 0.8 mM tBOOH for various times. Top, wild-type cells; bottom, cells expressing rck2-kd. Solid line, before addition of tBOOH; large-dashed line, 15 min after addition of tBOOH; dashed and dotted line, 30 min; small-dashed line, 60 min; and dotted line, 120 min.