Erythromycin-induced ribosome stalling and RNase J1-mediated mRNA processing in Bacillus subtilis

Abstract

Addition of erythromycin (Em) to a Bacillus subtilis strain carrying the ermC gene results in ribosome stalling in the ermC leader peptide coding sequence. Using ΔermC, a deletion derivative of ermC that specifies the 254 nucleotide ΔermC mRNA, we showed previously that ribosome stalling is concomitant with processing of ΔermC mRNA, generating a 209 nucleotide RNA whose 5′ end maps to codon 5 of the ΔermC coding sequence. Here we probed for peptidyl-tRNA to show that ribosome stalling occurs after incorporation of the amino acid specified by codon 9. Thus, cleavage upstream of codon 5 is not an example of 'A-site cleavage' that has been reported for Escherichia coli. Analysis of ΔermC mRNA processing in endoribonuclease mutant strains showed that this processing is RNase J1-dependent. ΔermC mRNA processing was inhibited by the presence of stable secondary structure at the 5′ end, demonstrating 5′-end dependence, and was shown to be a result of RNase J1 endonuclease activity, rather than 5′-to-3′ exonuclease activity. Examination of processing in derivatives of ΔermC that had codons inserted upstream of the ribosome stalling site revealed that Em-induced ribosome stalling can occur considerably further from the start codon than would be expected based on previous studies. © 2008 The Authors.