Fig. 2

The effect of intracistronic polarity during mboIIM2ΔA356 expression driven by the host RNAP. a Abolition of the polarity effect by reduction of mboIIM2ΔA356 gene length. Western blot of expressed mboIIM2 gene mutant variants in DH10B strain in the absence (lanes 1–4) and presence of λ phage N/nutL antitermination (lane 5). All cultures were 0.1% l-arabinose induced by 1 h at 37 °C. Equal amounts of total protein extracts from cultures harboring appropriate genes were analyzed after 12.5% SDS–PAGE and immunodetected with anti-M2.MboII antibodies. The schematic structure of genes was depicted below. Lane 6, molecular size markers (Thermo Scientific). Bands of the full-length (32 kDa) and short variants of M2.MboII (14.5 and 17.1 kDa) are marked by arrows. b Stability of mboIIM2 (white) and mboIIM2ΔA356 (black) gene transcripts analyzed by segmental real-time RT-qPCR. Results are shown as fold-change of expression. Error bars represent standard deviations from three replicates done twice. Location of primers used in RT-qPCR is depicted. c Suppression of polarity effect by N/nutL antitermination. The relative fold-change of particular segments of transcripts was measured by RT-qPCR and calculated as described in “Materials and methods” section. d Northern blot showing the stability pattern of mboIIM2 (WT), mboIIM2ΔA356 (Δ) and mboIIM2ΔA356Δ377 (ΔΔ) gene transcripts in the MC1061 wild-type and CH1828 rne-1 cells. MC1061 (lanes 1, 3 and 5) or CH1828 (lanes 2, 4 and 6) were transformed with appropriate plasmids and expression of mboIIM2 gene variants was induced with 0.1% l-arabinose for 20 min at 42 °C. Top panel: total RNA was subjected to 1.3% agarose-formaldehyde gel electrophoresis and then northern blot analyzed. The biotinylated probe against mboIIM2 internal sequence, and streptavidin-HRP and ECL chemiluminescent system detection were used. Positions of the full length (FL) and truncated (Trn) transcripts are marked. Bottom panel, ethidium bromide-stained 16S rRNAs were used as loading control. Lane 7, total RNA from non-mboIIM2 cells