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. 2017 Nov 23;6:e32038. doi: 10.7554/eLife.32038

Figure 4. TSS selection downstream and upstream of the modal TSS involves, respectively: increases and decreases in RNAP-dependent DNA unwinding.

(A) Use of single-molecule DNA nanomanipulation to define RNAP-dependent DNA unwinding. Single-molecule time traces with primers UGG, GGA, GAA, and AAU (positively supercoiled DNA in upper panel; negatively supercoiled DNA in lower panel). Rectangle with rounded corners highlights case of primer GGA, which programs TSS selection at position 7. Colors as in Figure 2B. (B) Transition-amplitude histograms (positively supercoiled DNA in upper panel; negatively supercoiled DNA in lower panel). (C) Differences in Δl¯obs,pos, Δl¯obs,neg, and DNA unwinding (bottom panel) with primers UGG, GGA, GAA, and AAU (means ± SEM).

Figure 4.

Figure 4—figure supplement 1. Single-molecule DNA-nanomanipulation: shorter DNA fragment enables detection of RNAP-dependent DNA unwinding with single-base-pair resolution.

Figure 4—figure supplement 1.

(A–B) Single-molecule time traces and transition-amplitude histograms with 2.0 kb DNA fragment [fragment length in previous work (Revyakin et al., 2004; Revyakin et al., 2005) and in Figure 2] (A), and with 1.3 kb DNA fragment (fragment length in Figure 4 and Figure 4—figure supplement 2) (B). Data for positively supercoiled DNA are at top; data for negatively supercoiled DNA are at bottom. Green points, raw data (30 frames/s); red points, averaged data (1 s window). (C) Mean transition amplitudes on positively supercoiled DNA (Δl¯obs,pos), mean transition amplitudes on negatively supercoiled DNA (Δl¯obs,neg), and DNA unwinding (means ± SEM). Use of 1.3 kb DNA segment provides SEM = 0.5 bp for data subsets of n = 30, sufficient for single-base-pair resolution.
Figure 4—figure supplement 2. Single-molecule DNA-nanomanipulation: analysis of primer-programmed TSS selection with primer GGA.

Figure 4—figure supplement 2.

Single-molecule time traces and transition-amplitude histograms with GGA, which programs TSS selection 7 bp downstream of the −10 element, and and with no primer, which also yields TSS selection 7 bp downstream of the −10 element (data for positively supercoiled DNA at top; data for negatively supercoiled DNA below). Primer GGA results in same RNAP-dependent DNA unwinding as in absence of primer.