FIG. 5.
Growth stage-specific RNAP shows temporal expression of in vitro phoPR promoter transcripts and the absence of PE2 with RNAP from a sigE mutant strain. (A) In vitro transcription of the phoPR promoter with RNAP isolated from stage T0, T3, and T4 cells grown in LPDM. The in vitro transcription reactions were carried out as described in Materials and Methods. M, RNA marker. In vitro transcripts were generated using RNAP from LPDM-grown MH5636 cells harvested at T0 (lane 1), T3 (lane 2), or T4 (lane 3) or from MH5654 (sigE) cells at T4 (lane 4). All reaction mixtures contained 5 pmol each of PhoP and *PhoR plus 1 mM ATP. (B) In vitro transcription products identified by primer extension. Markings and procedures were the same as for Fig. 4A, except the mRNA was generated by in vitro transcription (panel A, lanes 3 and 4).