Fig 8.
RusT employs a conserved sequence stretch for target recognition. (A) Base-pairing interactions between RusT and target mRNAs encoding transporters CCNA_02895, CCNA_03263, CCNA_01042, CCNA_00210, CCNA_03248, and ompW as predicted using the RNA hybrid and IntaRNA algorithms (56, 57). Nucleotide positions in the mRNA are marked relative to the start codon. The M1 (C53G) mutation in RusT and the corresponding M1 mutations in the mRNAs are indicated. (B) Regulation of target reporter fusions by RusT or RusT-M1. C. crescentus ΔvanAB ΔrusT cells carrying the indicated gfp reporter fusion in combination with either an empty control vector (pBV-MCS6; ctrl.), or the expression plasmid pPvan-RusT or pPvan-RusT-M1 were grown overnight in the presence of vanillate, and GFP expression was determined and quantified by fluorescence intensity measurements as described for Fig. 6. (C) CCNA_00210-3xFLAG protein expression in response to RusT overexpression. C. crescentus ΔvanAB CCNA_00210::3xFLAG cells carrying either an empty control vector [pBV-MCS6; (−)], or the expression plasmid pPvan-RusT (+) were grown to mid-exponential and early stationary phase (OD660 of 0.3 and 1.0, respectively) in the presence of vanillate. Total protein samples were analyzed by Western blot to determine CCNA_00210–3xFLAG levels. Expression of RNA polymerase (RNAP) was probed as loading control. Induction of RusT from pPvan-RusT was confirmed by Northern blot analysis; 5S rRNA served as loading control.