Fig. 1

Transcriptional slippage efficiency of E. coli and T7 phage RNA polymerases. a Details of the relevant sequence of the wild-type and mboIIM2ΔA356 single nucleotide deletion genes. The position of the A356 nucleotide is marked in red. Premature stop codon is underlined. b Level of expression of the mboIIM2ΔA356 deletion mutant in frame 0 (short product [Δ]—14.5 kDa) and after insertional slippage (full length [FL] product WT—32 kDa), respectively, determined by western blotting. Expression was driven by E. coli RNAP (DH10B, lanes 1–4) or T7 RNAP (ER2566, lanes 5–8) from a gene located on pBAD24/T7mboBΔ plasmid at 37 and 22 °C, respectively. Induction of expression was carried out for 3 h with 0.1% l-arabinose (lanes 2 and 4) or 1 mM IPTG (lanes 6 and 8), respectively. Lanes 1, 3, 5 and 7 cell lysates from non-induced cultures. Equal amounts of total protein extracts normalized to OD₆₀₀ were run on 12.5% SDS-PAGE, and immuno detected by using rabbit anti-M2.MboII polyclonal antibody and secondary anti-rabbit-AP. Note, that “leaky” production of M2.MboII proteins under non-inducing conditions (lanes 5 and 7) is due to lack of additional copy of lacI repressor gene on the pBAD24 backbone. Lane 9, molecular size prestained protein markers (EUR_x—Poland). c Immunodetection of the wild-type mboIIM2 gene product generated by the host (DH10B) or T7 RNAP (ER2566), respectively. Appropriate competent cells were transformed with pBAD24/T7mboWTlacI^q plasmid and the expression of the gene was induced for 2 h at 37 °C with 0.1% l-arabinose/0.05 mM IPTG (lane 2) or 1 mM IPTG (lane 4), respectively. Lanes 1 and 3, cell lysates from non-induced cultures; lane 5, prestained protein markers, including purified M2.MboII protein