Fig. 4: −10R, spacer length and tssR/−10LR determine G1p and G1d pauses in vitro.

Boxplots of pause strength for G1p (a) and G1d (b) pauses in the absence and presence of GreA or GreB. G1p (exuR, mraZ, ileX and mocA; n = 4 G1p promoters) and G1d (yieE, minC, gadW, mrdB and artP; n = 5 G1d promoters) promoters were used for the analysis (a–f). The pause strength was determined by dividing the signal intensity of run-off and paused RNA products to the signal intensity of paused RNA product in the gel for each in vitro template (Pause strength = Signal intensity[paused RNA]/(Signal intensity[paused RNA] + Signal intensity[run-off])). The pause strength in the absence of Gre factors was set to 1 (a, b). Boxplots show the effect on pause strength of −10R and tssR mutations in G1p promoters (c, f), and −10R and −10LR mutations in G1d promoters (d, e). Pause strength of the WT promoters was set to 1 (c–f). −10R (−10LR; tssR) Ri−/Ri+ , mutated −10R (−10LR; tssR) with decreased or increased Ri are indicated. The gray rectangle in each cartoon represents the motif used for mutation analysis. The original and mutated (colored in blue or red) DNA sequences designed to increase (Ri+) or decrease (Ri−) Ri are shown on the right in gene order. Two-tailed Mann-Whitney U-test was used for statistical analysis of the data. Effect of the spacer length on G1p (g) and G1d (h) pauses. The in vitro transcription was initiated on the WT template or on the mutant template with the shortened DNA spacer (left); different dinucleotide RNA primers overlapping the tssR were employed to alter the position of the TSS (right). The inset shows the run-off transcripts with higher exposure to visualize the faint bands. Data represent three independent experiments. Structural elements of the WT and mutated promoters are shown on the bottom. Each circle represents a single nucleotide. Open blue circles, −10R; Dark red circle, overlapped nucleotide between spacer and tssR/−10LR; Open black and dark red circles, spacer; Red circles, tssR; Red and orange circles, −10LR; Filled red circle, TSS. Red arrows indicate TSS. WT, wild-type promoter; SD2, spacer with 2-nt deletion; RPS, relative pause strength. The analysis included the data from two or more independent experiments.