Figure 5.

Lpm inhibits σ⁷⁰-dependent RNA synthesis from promoter-less templates. (A) The sequences of the minimal DNA scaffold and the annealed 3 nt RNA primer (pRNA3) are shown. Nucleotides are numbered with respect to the site of the first nucleotide addition (+1). (B) Synthesis of RNA initiated from pRNA3 by core RNAP (lanes 1–10) or holoenzyme (lanes 11–20) in the presence of increasing concentrations of Lpm. Transcription was initiated from either single-stranded template (ssDNA, lanes 1–5, 11–15) or assembled scaffold (scaffold, lanes 6–10, 16–20). The insert on the right shows the low exposure of the gel for lanes 16–20. The lengths of the RNA chains are indicated on the right. (C) Synthesis of RNA initiated from pRNA3 at the scaffold by the mutant holoenzyme or core RNAP^R337A in the presence of increasing concentrations of Lpm. (D) Quantification of the experiments shown in (B) and (C). The quantity of 9–12 nt RNA was plotted as a function of the inhibitor concentration. (E) The RNA primer length affects the sensitivity of RNA synthesis to Lpm. A scheme of the assembled scaffold and the 7 nt RNA primer (pRNA7) is shown. The panel on the right shows the RNA products produced after the addition of [³²P]-UTP to the RNA primers (pRNA3 and pRNA7) in the presence of Lpm. The length of the synthesized RNA is indicated.