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. 2019 Nov 27;10:5397. doi: 10.1038/s41467-019-13408-7

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — The cellular abundance of BrfA is negatively regulated by trans-translation. a A schematic representation of the brfA open reading frame, showing the intrinsic transcriptional terminator sequence within the coding region (indicated by “terminator” at the top and the wild type (WT) enlarged view below). Shown also are the corresponding sequences of the gfp-brfA(2–62)-flag and gfp-brfA(no_term)-flag constructs (see b), in which nucleotide substitutions and the FLAG tag are indicated by reverse and underline, respectively. b Schematic representations of the gfp-brfA-flag constructs with wild-type terminator sequence (gfp-brfA(2–71)-flag) and its derivatives with defective terminator signals. These gfp-brfA derivatives were placed under the constitutive mifM promoter. The lacZ gene is also placed downstream of the gfp-brfA derivatives. “SD” and asterisks indicate Shine-Dalgarno sequence and synonymous mutations, respectively. c Cellular accumulation of the products of the wild-type construct (lanes 1, 2) as well as the brfA(2–62) (lanes 3, 4) and the brfA(no_term) (lanes 5, 6) constructs. They were expressed in the smpB⁺ (odd numbers) or the ΔsmpB (even numbers) strains and analyzed by anti-GFP (upper) or anti-FLAG (lower) immunoblotting. d β-Galactosidase activities (mean ± s.d., n = 3) of the cells horbaring lacZ at the downstream of wild type (columns 1, 2), the brfA(2–62) (columns 3, 4), or the brfA(no_term) (columns 5, 6) derivatives of the gfp-brfA in the presence (odd numbers) or absence (even numbers) of smpB. “s.d.” indicates standard deviation. Source data are provided as a Source Data file.