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. 2015 Oct 1;43(21):10525–10533. doi: 10.1093/nar/gkv995

Figure 2.

Figure 2.

Effect of EF-G single site mutants on total protein synthesis and tRNA translocation. (A) Effect of overexpression of EF-G mutants on the growth of E.coli BL 21. Black arrow indicates the time that IPTG was added. (B and C) Effects of EF-G overexpression on total protein synthesis in vivo. Total soluble proteins from E.coli BL 21 overexpressing EF-G wt or mutants were analyzed by SDS-PAGE (B). After 35S-methionine addition, the ratio of proteins containing 35S-methionine increased over time as new proteins were continued to be synthesized in cells (C). (D) Brief illustration of the pyrene-modified mRNA translocation assay. The translocation action was monitored by tracing the fluorescence of pyrene attached to the 3′ end of the mRNA. The fluorescence emission is high (red star) before translocation and decreases (black star) upon translocation. (E) Relative fluorescence of PRE state ribosomes programmed by the labeled mRNA with incorporation of EF-Gwt or mutants. Error bars, s.e.m. (n = 3 technical replicates). ***P < 0.001.