Expression of the bmr operon is extinguished at the post-transcriptional level by Rae1 cleavage downstream from the attenuator. (A) Annotated structure of the bmr operon. ORFs are represented by gray boxes, the promoter by a rightward-pointing arrow, and the transcription attenuator (att) and terminator (ter) by hairpin structures. The two primary transcripts (bmrB and bmBCD) and the processed mRNA (bmrCD) are shown as wavy lines and their approximate sizes are indicated. The position of the probes used is indicated by black bars. (B) Northern blot showing levels of bmr and abrB mRNAs at times after rifampicin addition in WT and Δrae1 strains at mid-log phase. (C) Northern blot showing levels of bmr mRNAs at times after rifampicin addition to the Δrae1 strain containing either a plasmid expressing WT Rae1 (pRae1) or the catalytic mutant (pRae1D7ND81N) at mid-log phase. Blots were probed in a sequential order with oligo CC2344 (bmrC) (B,C), oligo CC2145 (abrB) (B), a riboprobe against bmrB (B,C), and finally with a probe complementary to 16S rRNA (CC058) as a loading control (B,C). (D) Primer extension assay on total RNA isolated from WT and strains lacking Rae1 (Δrae1), RNase J1 (ΔrnjA), and both (Δrae1 ΔrnjA) using oligonucleotide (CC2344) that hybridizes to the early bmrC coding sequence. Sequence lanes are labeled as their reverse complement to facilitate direct read-out. The mapped 5′ ends are shown to the right of the autoradiogram. A reverse transcriptase (RT) stop corresponding to the attenuator (att) is indicated. This experiment was repeated twice. (E) Predicted secondary structure (Mulfold: http://www.unafold.org/mfold/applications/rna-folding-form.php) of the bmrB–bmrC intergenic region. Coordinates are given relative to the AUG start codon of bmrC (boxed). The two 5′ extremities mapped by RT are shown. The predicted attenuator stem–loop (att) is shown and the Shine–Dalgarno (SD) sequence of B. subtilis 16S rRNA is underlined. The start and the stop codon of the predicted bmrX ORF are indicated.
