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. 2024 Aug 1;52(18):11234–11253. doi: 10.1093/nar/gkae629

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© The Author(s) 2024. 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 (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 6. — Target mRNA expression and translation do not affect mRNA editing. (A) Effect of mRNA abundance on mRNA editing of amyA (upper panels) and fakB2 (lower panels). The AHT-inducible P_tet promoter cassette was integrated upstream of two target gene operons (upper scheme), malX-amyBA-malCDA (EC3456, upper panels) and fakB2-SPy_1492-SPy_1491 operons (EC3459, lower panels), respectively. Strains were grown in the presence of AHT (0–100 ng/ml) until mid-logarithmic phase. mRNA expression levels were measured by qRT-PCR with gyrA and ropB as reference genes (left panels), and editing levels were examined by Sanger sequencing (right panels). Editing and expression were normalised to the respective mean at 0 ng/ml AHT for each strain. Statistical analysis was performed using one-way ANOVA and Dunnett's post-hoc test. (B) Effect of translation on mRNA editing. The ermB regulatory region including the first 30 nt of ermB (shades of blue) was fused to an artificial, in-frame A-to-I editing site (red) and the firefly luciferase ffluc (yellow). Addition of erythromycin (Erm) causes ribosome stalling on the ermB leader peptide but induces translation of the ermB’-ffluc fusion (upper scheme). S. pyogenes SF370 was transformed with the Erm-inducible wildtype reporter (pEC3045), an erythromycin insensitive (off, pEC3046) and a constitutively high translation control (on, pEC3047), and cells were exposed to different concentrations of erythromycin for 30 min at mid-logarithmic growth phase. Luminescence was measured and expressed as log₁₀-transformed relative luminescence units (RLU) per OD (upper left panel), and editing levels were examined by Sanger sequencing (lower left panel). As an additional control, S. pyogenes SF370 was transformed with the constitutively high translation control reporter (on, pEC3047) and its start codon mutant version (on^STOP, pEC3069), and cells were treated and analysed as before (right panels). Statistical analysis was performed using one-way ANOVA and Dunnett's post-hoc test (left panels), or unpaired two-sided t-test (right panels). (A, B) *** P < 0.001; ** P < 0.01; * P < 0.05; ns, not significant.