Figure 4.

Overexpression of lon and rpoH induces A-site mRNA cleavage. (A) Overexpression of lon induces A-site mRNA cleavage. flag-(m)ybeL-PP transcripts were expressed in ssrA⁻ Δrnb backgrounds at 37°C. Where indicated, the lon or lon(S679A) genes were overexpressed from a plasmid-borne arabinose-inducible promoter. (B) Lon immunoblot analysis. Urea-soluble protein was isolated from cells of the indicated genotype that had been cultures at 30°C, 37°C or 42°C for 2.5 h. The bottom panel shows Lon levels in ssrA⁻ Δrnb cells that had been cultured at 30°C for 1.5 h, then shifted to 42°C for the indicated number of minutes. (C) Overexpression of rpoH induces A-site mRNA cleavage. flag-(m)ybeL-PP transcripts were expressed in the indicated genetic backgrounds at 37°C. Where indicated (+), rpoH was overexpressed from a plasmid-borne arabinose-inducible promoter. In (A and C), the migration position of flag-(m)ybeL-PP transcript that is truncated at the stop codon is indicated, and horizontal arrows indicate an additional yoeB-dependent transcript. (D) Immunoblot analysis of Lon. Urea-soluble protein was isolated from cells of the indicated genotype that had been grown at 37°C. Where indicated (+), the σ³² heat-shock transcription factor (rpoH) was overexpressed. Samples were analyzed by immunoblot using polyclonal antisera to Lon protease.