Fig. 3 | Nature Communications

Fig. 3

From: Activity of acetyltransferase toxins involved in Salmonella persister formation during macrophage infection

Fig. 3

Tac toxins block translation through acetylation of aminoacyl-tRNAs. a Cell-free expression assays leading to the production of the control protein DHFR (red asterisk) from template DNA without toxins or with purified TacT, TacT2STm, TacT2SEn or TacT3, added from the onset of the assay. [14C]Ac-CoA was added to all the samples. All samples were analysed by SDS-PAGE, and the production of DHFR was revealed by Coomassie staining (CM). tRNAs extracted from the samples were analysed by acid-urea PAGE and revealed by methylene blue staining (MB) and acetylation tracked by autoradiography (AR). Equivalent amount of toxins added to the sample was tested by western blotting against His tag of the toxins (WB). Quantification of inhibition of the production of DHFR (spotted bars) and acetylation of tRNAs (dashed bars) in all conditions is reported in the bar charts, where data represent the mean ± SEM (n ≥ 3). b Surface of the Ac-CoA-binding site of TacT3 with Ac-CoA represented as solid sticks for the orientation in TacT3 and shadowed for the orientation of Ac-CoA in TacT. Trp142 is in yellow and Gly145 is in red. c Exposure of tRNA molecules acetylated by TacTs to Pth treatment in vitro. tRNA molecules acetylated by different TacTs in vitro were subsequently incubated with purified Pth, acetylation was assessed by autoradiography, before or after samples were treated with purified Pth. All the samples were supplemented with [14C]Ac-CoA. Treated tRNA molecules were separated on acid-urea polyacrylamide gel and revealed by methylene blue staining (MB) (top panel). Acetylation was tracked by autoradiography (AR) (lower panel). d Model of activity of Tac toxins

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