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. 2018 Jan 30;46(4):1973–1983. doi: 10.1093/nar/gky023

Figure 2.

Figure 2.

Allosteric regulator pppGpp and substrate GDP synergize for maximum enzymatic activity of E. coli RelA. Enzymatic activity of 30 nM RelA assayed in the presence of 1 mM (A-C) or 4 mM (D-F) ATP and 0.3 mM of either 3H-labelled GTP (A) or GDP (B and C); in the presence of equal amounts of GDP and GTP (0.3 mM each) (D and E); or in the presence of 0.2 mM GDP and 1.1 mM GTP (F). In the case of D–F both 3H-ppGpp and 3H-pppGpp were synthesized in the reaction. As indicated on the figure, the reaction mixtures were supplemented with combinations of 0.5 μM vacant 70S or ‘starved’ ribosomal complexes programmed with 2 μM model mRNA(MF) encoding Met-Phe and deacylated tRNAiMet (2 μM; P-site) and tRNAPhe (2 μM; A-site) as well as ppGpp or pppGpp added either 100 μM (A and B) or varying concentrations (C). All experiments were performed in HEPES:Polymix buffer, pH 7.5 at 37°C in the presence of 5 mM Mg2+. Error bars represent SDs of the turnover estimates by linear regression and each experiment was performed at least three times.