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 DeltaermC, a deletion derivative of ermC that specifies the 254 nucleotide DeltaermC mRNA, we showed previously that ribosome stalling is concomitant with processing of DeltaermC mRNA, generating a 209 nucleotide RNA whose 5' end maps to codon 5 of the DeltaermC 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 DeltaermC mRNA processing in endoribonuclease mutant strains showed that this processing is RNase J1-dependent. DeltaermC 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 DeltaermC 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.
Yao
Shiyi
S
Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine of New York University, New York, NY 10029, USA.
Blaustein
Joshua B
JB
Bechhofer
David H
DH
D001412:Bacillus subtilis / Q000187:drug effects* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D015152:Blotting, Northern; D004917:Erythromycin / Q000494:pharmacology*; D008780:Methyltransferases / Q000235:genetics; D009690:Nucleic Acid Conformation; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D012323:RNA Processing, Post-Transcriptional; D012329:RNA, Bacterial / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D012343:RNA, Transfer / Q000032:analysis; D012260:Ribonucleases / Q000378:metabolism*; D012270:Ribosomes / Q000187:drug effects*; D017384:Sequence Deletion
D001426:Bacterial Proteins; D011500:Protein Synthesis Inhibitors; D012329:RNA, Bacterial; D012333:RNA, Messenger; D004917:Erythromycin; D012343:RNA, Transfer; D008780:Methyltransferases; C035181:rRNA (adenosine-O-2'-)methyltransferase; D012260:Ribonucleases