Fig. 3: Characterization of factors involved in pep-tRNA drop-off in the early elongation stage.
From: Quality control of protein synthesis in the early elongation stage
a Depletion of RF3 (encoded by prfC) alleviates the temperature sensitivity of the pthts strain. WT, ΔprfC, pthts, and ΔprfC/pthts strains were serially diluted (1:10), spotted onto LB plates, and cultivated overnight at the indicated temperatures (30, 37, 39, and 43 °C). b Overall change in aa-tRNAs (left panel) and dipep-tRNAs (right panel) in pthts (blue) and ΔprfC/pthts cells (red) after the temperature shift to 43 °C. The calculation method of each relative value is described in Methods. Data are presented as means±s.d. of three independent experiments. c Overexpression of RRF aggravates the temperature sensitivity of the pthts strain. pthts cells transformed with empty vector (empty) or expression vector for frr (pRRF) were serially diluted (1:10), spotted onto LB plates containing the indicated concentrations of arabinose (0, 0.1, and 1 mM), and cultivated overnight at the indicated temperatures. d Overall change in aa-tRNAs (left panel) and dipep-tRNAs (right panel) in the pthts strain that expresses different levels of RRF. The cells were cultured in the presence of 0 mM (blue), 0.1 mM (green), and 1 mM (red) arabinose at 30 °C, and then shifted to 43 °C and sampled every 2 min for 14 min. The calculation method of each relative value is described in Methods. e Temperature sensitivity of the frrts strain. WT, pthts, and frrts strains were serially diluted (1:10), spotted onto LB plates, and cultivated overnight (the time is longer than that of (a)) at the indicated temperatures (30, 35, 37, 39, and 43 °C). The pictures of the spots of WT and pthts strains are shared with Fig. 1b. f Metagene analyses of RPFs mapped around the translation initiation sites of E. coli ORFs for WT (replicate 1, blue and replicate 2, pale blue) and frrts (replicate 1, orange and replicate 2, pale orange) strains incubated at 43 °C for 30 min. The positions of ribosome containing di-, tri-, and tetra-pep-tRNAs on P-site are indicated based on the distance between the P-site and the translation start site. Source data are provided as a Source Data file.
