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. 2017 May 18;45(12):7474–7486. doi: 10.1093/nar/gkx434

Figure 4.

Figure 4.

A defined mRNA region is used for the control of Rho-dependent transcription termination. (A) Schematic representing thiM ORF comprising codons 9–40. Nucleotides highlighted in gray and in black represent Rho termination sites and the 201–210 nt region, respectively. Mutations and deletions (Δ) are shown in the structure. (B) In vitro transcriptions performed using wild-type thiM mRNA. Transcriptions were done in the absence (−) or presence (+) of 50 nM NusG or 50 nM Rho. Read-through and termination transcripts are indicated at the right. Termination efficiencies are indicated below. Note that a read-through product of 400 nt is expected. (C) Sequence analysis of cytosine (C%) and guanine (G%) distribution in the thiM sequence. A scanning window of 25 nt was used to determine C and G occurrences as a function of the transcription start site (TSS). The shaded region represents the position of the stem-loop. (D) RNase H probing of Rho binding on thiM mRNA. RNase H assays were performed in the absence (−) or presence (+) of 50 nM Rho. Cleavage assays were done using DNA oligonucleotides targeting regions 130–139 (130) or 201–210 (201). Cleavage products and cleavage efficiencies are indicated on the right and below, respectively. Non-cleaved transcripts (N) are shown as controls. (E) β-Galactosidase assays of transcriptional thiM–lacZ fusions performed in the absence or presence of 500 μg/ml TPP. Measurements were performed for mutants targeting the C-rich region (M1–M3, M6 and M7) and the stem-loop (M4 and M5). Values were normalized to the activity obtained in the absence of TPP. The average values of three independent experiments with SDs are shown. (F) β-Galactosidase assays of transcriptional thiM–lacZ fusions of M2, M6 and M7 mutants in the context of various sizes of thiM open reading frame. Enzymatic activities were conducted in the absence or presence of 500 μg/ml TPP. Values were normalized to the enzymatic activity obtained in the absence of TPP. The average values of three independent experiments with SDs are shown.