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. 2019 Apr 17;47(12):6396–6410. doi: 10.1093/nar/gkz274

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© The Author(s) 2019. 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 Non-Commercial 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 1. — Ectopic overproduction of the attenuator sRNA rnTrpL decreases the level of the polycistronic trpDC mRNA in S. meliloti. (A) Schematic representation of the three trp operons and the attenuator RNA rnTrpL. ORFs are depicted as gray arrows, the RpoD-like promoter is shown as a gray rectangle, a transcription start site is indicated by a flexed arrow and a transcription terminator by a hairpin (according to 23 and 59). The terminated attenuator sRNA rnTrpL (110 nt; synonym RcsR1) is shown as a white box with start and stop codons indicated. Below this box, regions corresponding to stem-loops (SL) 1, 2 and 3 are indicated by convergent arrows. SL2 corresponds to the antiterminator and SL3 to the terminator of transcription. SL2 is preferentially formed in the presence of translating ribosomes that pause upon tRNA^Trp shortage. For the rnTrpL secondary structure, see Figure 2. The proposed binding site of rnTrpL in the trpD ORF is indicated by a white box. (B) Schematic representation of the plasmid pRK-rnTrpL. Transcription of rnTrpL from this plasmid is expected to be terminated at SL3 in TY medium (see Supplementary Figure S1). (C) Northern blot analysis of RNA isolated from the EVC strain 2011 (pRK4352) and the overexpressing strain 2011 (pRK-rnTrpL), grown in TY. The membrane was first hybridized with an rnTrpL-specific probe and subsequently hybridized with a 5S rRNA-specific probe (loading control). (D) Results of qRT-PCR analyses of the indicated mRNAs of the trpDC operon and trpE (control mRNA). Strain 2011 (pRK-rnTrpL) was compared to the EVC and log₂(fold change) (log₂FC) in mRNA steady-state levels were calculated. Shown are the results from three independent experiments, each performed in technical duplicates (mean values and standard deviations are indicated).