Analysis of transcription of sgrS-S and sgrS-S-LS2 directed by the tac promoter. (A) DNA sequences of the chimeric Ptac-sgrS-S and Ptac-sgrS-S-LS2 genes. The sequence corresponding to sgrS-S is shown as regular letters, whereas the tac promoter sequence is shown as italic letters. The sequences of the −10 and −35 regions of the tac promoter are boxed. The inverted repeat sequences of the sgrS-S terminator are indicated by horizontal arrows. The inserted sequences to stabilize the terminator stem are shown as bold letters. (B) In vivo expression of the Ptac-sgrS-S and Ptac-sgrS-S-LS2. TM772 (ΔsgrS Δhfq) cells harboring indicated plasmids were grown in LB medium. Total RNAs were prepared at A600 = 0.4, and 20 µg of RNA samples was subjected to Northern blotting using the SgrS-S probe. Arrowheads represent the full-length transcripts. (C) SDS-PAGE analysis of the purified RNAP-Flag. The affinity-purified PNAP-Flag sample (5 µL) was analyzed by SDS-PAGE and Coomassie staining as described in Materials and Methods. The bands corresponding to RNAP subunits are indicated on the left. Protein size markers are shown on the right. (D) In vitro transcription of the Ptac-sgrS-S and Ptac-sgrS-S-LS2. The transcription reaction was performed as described in Materials and Methods. The samples were subjected to Northern blotting using the SgrS-S probe. Arrowheads represent the full-length transcripts.
