Figure 5. EVN arrests the ribosome translating the EmtAL peptide in vivo.

a, Schematics of the emtAL-ermCL-ermC-rfp reporter for testing EVN-dependent translation arrest within the emtAL leader ORF VFL sequence. In the wt ermCL-ermC operon, macrolide dependent ribosome stalling at the 9^th codon of ermCL leads to activation of ermC expression^25,47. In the shown construct, the first 10 ermCL codons were replaced with 7 codons of emtAL to render reporter activation by ribosome stalling at the Phe₆ codon. The reporter expression is monitored following fluorescence of the RFP protein, whose gene, lacking its own start codon, is fused in frame after the first four codons of ermC. The nucleotide sequences of the hybrid emtAL-ermCL leader ORF and the encoded protein are shown. The EVN stalling motif VFL of EmtAL is boxed in red. b, Drop-diffusion test demonstrating activation of the reporter expression due to EVN-dependent ribosome stalling. A drop of antibiotic solution was placed on agar plates inoculated with E. coli cells carrying the reporter cassette. Diffusion generates an antibiotic concentration gradient preventing cell growth near the site of application (black area). Farther away from the drop, subinhibitory concentrations of EVN (but not of the non-inducing control antibiotic spectinomycin, SPC) activate the reporter expression (left). Changing the EmtAL’s VFL motif to VFA diminishes the EVN-mediated induction of RFP expression (right). c, Induction of the reporter expression monitored in liquid culture in cells grown at varying concentrations of EVN or SPC. The dots on the plot represent the average experimental values between three biological replicates with the error bars indicating s.e.m.