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. 2022 Jun 14;50(11):6174–6189. doi: 10.1093/nar/gkac497

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© The Author(s) 2022. 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 5. — Lincomycin-dependent induction of VmlR expression is caused by ribosomal stalling at the uORF(MIN). (A) The effects of truncations/mutations in stem loops SL1, SL3 and SL4 on lincomycin-dependent induction of VmlR expression. SL-1 is essential for lincomycin-dependent induction of VmlR expression and SL-3 is crucial for repression of VmlR expression in the absence of the inducer. Wild-type (VHB570), ΔSL-1 (VHB575), ΔSL-3 (VHB474), SL-3* (VHB572), SL-3* ΔSL-1 (VHB583) and SL-4* (VHB594) cells were treated with lincomycin for 30 min and VmlR_His6 expression was detected by immunoblotting. Mutation sites of SL-3* and SL-4* are in red. (B) 30S ribosomal toeprint of the uORF(MIN). Toeprints were conducted using wild-type (wt) and Shine-Dalgarno sequence mutant (SD uORF*) templates as indicated. Reactions were carried out in the absence (–) or presence (+) of B. subtilis 30S ribosomal subunits. All reactions contained E. coli fMet-tRNA_i^fMet. The start codon (AUG), stop codon (UAA), the position of the 30S ribosomal subunit-dependent toeprint located 16 nucleotides downstream the A residue of the start codon, as well as the full-length reverse transcription product (FL) are marked. Sequencing lanes (A, C, G and U) are shown. (C) Translation of the uORF(MIN) is essential for lincomycin-dependent induction of VmlR expression. MIC for lincomycin for wild-type (VHB570), SD uORF* (VHB573) and uORF* (VHB604) strains are indicated in purple. Mutation sites of SD uORF* and uORF* are in red. All experiments were performed at least twice yielding similar results. Expression of VmlR_His6 was assessed by α-His₆ immunoblotting. (D) uORF sequence variants from other Bacillus species are competent in induction of vmlR expression in a lincomycin-dependent manner. Wild-type B. subtilis uORF(MIN) (VHB570), MDN (VHB839), MDK (VHB840) and MRK (VHB841) uORF variant cells were treated with lincomycin for 30 min and expression of VmlR_His6 was detected by α-His₆ immunoblotting.