Skip to main content
. 2005 Mar 31;24(8):1584–1595. doi: 10.1038/sj.emboj.7600636

Figure 8.

Figure 8

Ribosomes form stable complexes with nonstop mRNA. (A) YS091 (dcp1-2 ski7Δ) cells harboring pIT765 (pGAL1p-HIS3-His6, HIS3, left panels) or pIT766 (pGAL1p-his3-His6-ns, nonstopHIS3, right panels) were grown on SG-UraMet at 25°C. After incubation at 37°C for 90 min, cells were harvested in the presence (top panels) or absence (bottom panels) of CHX. Cell extracts were resolved by velocity sedimentation on 10–50% sucrose gradients. RNA samples prepared from the indicated fractions were analyzed as shown in Figure 3A. (B) YS091 (dcp1-2 ski7Δ) cells harboring pIT826 (pGAL1p-FLAG-HIS3, WT) or pIT827 (pGAL1p-FLAG-his3-ns, NS) were grown on SG-Ura medium at 25°C. After incubation at 37°C for 90 min, cells were labeled with [35S]methionine for 10 min followed by immunoprecipitation. Total cell extracts (lanes 1 and 2) and immunoprecipitated samples (lanes 3 and 4) were subjected to SDS–PAGE and visualized by autoradiography. (C) W303 cells harboring pIT922 (pGPDp-FLAG-HIS3, left panel) or pIT923 (pGPDp-FLAG-his3-ns, right panel) were grown on SC-Ura medium at 30°C. After incubation in medium without glucose for 10 min, cells were harvested. Sample preparation and hybridization were performed as described in Figure 3A.