Skip to main content
. 2018 Aug 27;46(20):10969–10982. doi: 10.1093/nar/gky765

Figure 5.

Figure 5.

ChvR interacts with two independent sites of chvT mRNA. (A) Schematic representation of the translational chvT::gfp fusions (under control of the constitutive PrsaA promoter). Reporters comprise the 5′ untranslated region plus the first 45 nucleotides of the chvT CDS. Location of ChvR binding sites 1 and 2 are indicated by blue boxes, positions of single nucleotide exchanges are marked in red. (B) Partial repression of chvT::gfp by ChvR via binding site 1. C. crescentus ΔvanAB carrying the indicated gfp reporter fusion were co-transformed with either a control plasmid (pBVMCS6), or a plasmid overexpressing ChvR or ChvR-M1 under the control of the vanillate-inducible promoter Pvan (pKF382-1 or pKF395-1, respectively) were grown overnight in PYE supplemented with vanillate. GFP fluorescence was determined by plate reader measurements. For each fusion, GFP levels in the presence of the control plasmid were set to 1, and relative changes were determined for cells expressing ChvR. GFP levels were calculated from three biological replicates; error bars indicate the standard deviation. (C, D) Secondary structures of ChvR and the 5′ UTR of chvT mRNA (from the transcriptional start site to the start codon) based on bioinformatic predictions (45). (E) Predicted base-pairing interactions forming between ChvR and chvT mRNA. Both interactions at binding site 1 (base-pairing of the second stem-loop of ChvR (nts 31–42) and chvT mRNA (nts −70 to −59 relative to the translational start site)) and at binding site 2 (base-pairing of the third stem-loop of ChvR (nts 63–73) and chvT mRNA (nts −20 to −10 relative to the translational start site). Positions of single-nucleotide exchanges generating the compensatory mutants M1 and M2 are indicated. Expression of all ChvR variants was confirmed by Northern blot analysis (Supplementary Figure S7B). (F-G) Analysis of GFP fluorescence of C. crescentus ΔvanAB carrying the indicated gfp reporter fusion in combination with either a control construct, or plasmids overexpressing ChvR, ChvR-M1, ChvR-M2 (pKF414-1) or ChvR-M1M2 (pKF418-1). Experimental details as in (B).