Figure 3
Mapping of the promoter identified upstream of secG (P secG ). (a) Primer extension using 5 μg of total RNA extracted from the wild type at 15°C and a 5’-end-labeled primer specific for the 5’region of secG (rnm014). The arrow indicates the fragment extended with this primer (128bp). ATCG lanes are sequencing ladders obtained with M13 DNA and a specific radiolabeled primer, which allowed us by size comparison of the unknown product with the ladder to determine the first nucleotide at the 5’-end of secG mRNA. (b) RACE to map the 5’-end of the secG transcript. Reverse transcription was carried out on 6 μg of total RNA extracted from RNase R- strain, using a secG specific primer (smd039). PCR signals upon treatment with TAP (lane T+) or without treatment (lane T-) were separated in a 3 % agarose gel. The arrow indicates the signal upon TAP treatment, which corresponds to a primary transcript. Molecular weight marker (Hyperladder I - Bioline) is shown on the left. (c) Sequence of the secG promoter (PsecG). The nucleotide corresponding to +1, as determined by primer extension and 5’ RACE, is shown in bold. The −35 and −10 boxes are underlined, and the ATG start codon of secG is indicated by a box.