Figure 4

Primer extension and 5’ RACE analysis of the rnr genomic region. (a) Primer extension was carried out with 5 μg of total RNA extracted from the RNase R^- strain at 15°C, using a 5’-end-labeled primer specific for the 5’region of smpB (rnm002). The arrows indicate the fragments (a – 188bp, b – 182bp) extended from this primer. The comparison of the fragments sizes with the reading of a generated M13 sequencing reaction provided the determination of the 5’-end of the obtained mRNAs. (b) 5’ RACE mapping of the smpB transcript. Reverse transcription was carried out on 6 μg of total RNA extracted from wild type and mutant derivatives as indicated on top, using an smpB specific primer (rnm011). PCR signals upon treatment with TAP (lane T+) or without treatment (lane T-) were separated in a 3 % agarose gel. As a negative control, the same experiments were performed in the SmpB^- strain. The arrows indicate the specific 5’ RACE products (1, 2). Molecular weight marker (Hyperladder - Bioline) is shown on the left. (c) Sequence of the region that comprises the 3’end of rnr and the 5’end of smpB. The nucleotides corresponding to the 5’-end of the extended fragments or to the 5’ RACE products are highlighted in bold. Letters (a, b) or numbers (1, 2) denote primer extension or 5’ RACE results, respectively. The ATG of smpB and the stop codon of rnr (TAA) are delimited by a dashed box and the putative RBS is indicated.