Fig 5. Permissive ppGpp-mediated inhibition of mRNA translation.

(A) Scheme of the translating 70S complex. (B) In vitro translation of mTufA and mInfA derivatives harboring a coding sequence for the Lumio labelling system (Experimental conditions and analysis). Protein synthesis reactions were started in the presence of 2 mM GTP and increasing concentrations of ppGpp. Synthetized proteins were fluorescently labelled and resolved by 20% SDS-PAGE. The resulting images were analyzed by pixel densitometry using ImageJ [37] to estimate translation efficiencies (TEs). (C) Log of ppGpp concentrations was plotted and used for determining the inhibitory concentration for 50% inhibition (IC₅₀) using a same-site competition model. (D) Comparison of inhibitory constants for both mRNAs as measured during 30S IC formation (white) or overall translation efficiency (gray). Mean and standard deviations from three measurements are plotted (S1 Data). GTP, guanosine triphosphate; mInfA, InfA mRNA; mTufA, TufA mRNA; TE, translation efficiency.