FIGURE 5.

Transcription termination and gene control by the tandem TPP riboswitch from the tenA mRNA of B. anthracis. (A) In vitro transcription of the 5′ UTR of the B. anthracis tenA mRNA. Bands labeled T1, T2, and FL correspond to RNA products terminated at the first expression platform, the second expression platform, and full-length transcription, respectively. (B) Line plots comparing the amounts of RNA transcripts produced during in vitro transcription after 30 min incubation with or without added TPP, as indicated. Data were derived from the PAGE image in A, where the top of the gel corresponds to the right of the line plot. (C) Plot of the amount of β-galactosidase reporter gene expression resulting from fusions to several versions of the B. anthracis tenA mRNA 5′ UTR. The Thi1+T1 construct carries only the first riboswitch. Mut1 (G41C, A42U) and Mut2 (G210C, A211U) constructs carry mutations in the junction between P3 and P4 in the first or second aptamer (Fig. 3) that disrupt TPP binding (data not shown; Winkler et al. 2002b). Mut1-Mut2 construct carries both sets of disruptive mutations. Constructs were tested in transformed B. subtilis cells in minimal medium supplemented with various concentrations of thiamine as indicated.