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. 2021 Jul 13;49(14):8384–8395. doi: 10.1093/nar/gkab608

Figure 3.

Figure 3.

Tre23 ADP-ribosylates the 23S RNA of actively translating ribosomes. (A and B) Native PAGE (A) and SDS-PAGE (B) analyses of in vitro couple translation-transcription reaction supplemented with 6-biotin-17-NAD+ (biotNAD+) and with Tre23, as indicated. Samples were stained with Coomassie blue (left panels) or detected using streptavidin-AP (right panels). Mass spectrometry analysis of the indicated band identified subunits of 30S and 50S ribosome (Ribos.). (C) RNA extracted from of in vitro transcription-translation reactions supplemented with 6-biotin-17-NAD+ (biotNAD+), Tre23 and Tri23, as indicated were stained with ethidium bromide (EtBr, left panel) or detected using streptavidin-AP conjugate (right panel). The positions of tRNAs, 16S and 23S ribosomal RNAs are indicated. (D) Western-blot analyses with anti-Strep antibodies of GFP-strepII in vitro transcription-translation in presence of ribosomes purified from wild-type E. coli cells carrying empty pNDM220 vector (Ø), or cells producing Tre23. Ribosomes (R) included in the coupled transcription-translation kit were used as control. Ribosome profile changes over time after toxin induction are shown in Supplementary Figure S2. (E) RNA analyses of in vitro transcription-translation reactions supplemented with 6-biotin-17-NAD+ (biotNAD+), and Tre23, as indicated, using ribosomes purified from wild-type E. coli cells bearing empty vector (Ø), or cells producing Tre23. RNAs were stained with EtBr (left panel) or detected using streptavidin-AP (right panel). (F) EtBr staining (left panel) and Streptavidin-AP (right panel) RNA analyses of purified ribosomal RNAs (rRNA), or purified ribosomes alone or supplemented with translation factors (F) or translation reagents (tRNAs, amino acids, nucleotides; A), in presence of 6-biotin-17-NAD+ (biotNAD+) and Tre23, as indicated.