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. 2016 Oct 25;45(8):4708–4721. doi: 10.1093/nar/gkw1018

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© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 6. — RtcB restores the ability of the ribosomes to translate canonical mRNAs in vivo. (A) Schematic depiction of the construct encoded by plasmid pACA-RNA43^SD used to ectopically express the rrsB^SD fragment that can be processed by MazF at the ACA-site to generate the mature RNA43^SD. Below, the control construct comprising the rrsB^SD fragment without the ACA-cleavage site is shown (pACA-RNA43^SD), which cannot be processed by MazF. The binding site of the primer S19 used for RT-PCR analysis shown in D is indicated. See text for details. (B) Scheme showing the corresponding gfp^aSD reporter gene of plasmid pgfp^aSD, which does not harbor ACA-sites not to interfere with mRNA levels due to activation of MazF during stress treatment (Oron-Gottesman et al., submitted). See text for details. (C) The relative fluorescence units (RFU, normalized to OD₆₀₀) were measured for strains MG1655 and the isogenic rtcB-deletion strain harboring the gfp^aSD reporter gene and either plasmid pACA-RNA43^SD or plasmid pGCA-RNA43^SD at the indicated time points before (0 min) and during (30 and 60 min) recovery from SHX treatment. The experiment was performed in triplicate; error bars indicate the standard deviation from the mean (***P < 0.001). (D) RT-PCR analysis to monitor the formation of the 16S^SD rRNA by RtcB-mediated ligation during recovery from SHX-treatment in strains MG1655 and MG1655ΔrtcB. Primer pair S7/S19 specifically amplifies a 182 nts long product only after ligation of the 16S^Δ43 rRNA and the ectopically expressed RNA43^SD (indicated by an asterisk). Primers S7/X15 served as internal control. NTC: no template control. The length of the marker bands in nts is given to the left.