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

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 Delta ermC, a deletion derivative of ermC that specifies the 254 nucleotide Delta ermC mRNA, we showed previously that ribosome stalling is concomitant with processing of Delta ermC mRNA, generating a 209 nucleotide RNA whose 5' end maps to codon 5 of the Delta ermC 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 Delta ermC mRNA processing in endoribonuclease mutant strains showed that this processing is RNase J1-dependent. Delta ermC 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 Delta ermC 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.