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. 2020 Nov 24;48(21):12336–12347. doi: 10.1093/nar/gkaa1139

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© The Author(s) 2020. 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/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 4. — Rescue of 30S-IC formation and ribosome binding by the double-mutant G57C / C74G. (A) (Left) Secondary structure model of the 5′-end pseudoknot in wild-type +42 tisB mRNA. Nucleotides forming stem 2 are indicated in grey and black boxes (left and right arms of the helix, respectively), and the G57 is highlighted by an orange circle. (Right) Representation of the stem 2 helix with the G57C base substitution (green base), C74G (red) and the double compensatory mutant Comp. 1 (purple). Disruption of Watson-Crick base pairing is indicated (red cross). (B) Toeprint assays on +42 tisB mRNA (WT) and mutated variants G57C, C74G, and the compensatory double mutant G57C/C74G (Comp. 1). The position of the toeprint signal at +15 is indicated. (C) Fluorescence anisotropy change of 5′-end FAM-labeled +42 tisB mRNA was conducted as in Figure 2B. Complexes were competed with a 50-fold molar excess of wild-type +42 tisB mRNA or the corresponding G57C, C74G, and G57C/C74G (Comp. 1) variants. *P-value < 0.05 (triplicate)