(A, B) Nucleic-acid scaffolds that enable EC reconstitution just upstream of sequences equivalent to the C346 H-NS-stimulated pause (A) and the C370 pause not stimulated by H-NS (B). Lowercase RNA, nt added by RNAP after reconstitution. *, position of [32P]CMP incorporation (red). Green, 3′ nt at the pause. (C, D) Pause assay reaction schemes (see ‘Materials and methods’). For assays on the fly, 10 μM CTP, UTP, and GTP (for ECC346) or CTP, ATP, and GTP (for ECC370) were added to ECs upstream from the pause at either 37°C or 20°C. For C346 delay assay (C), 10 μM CTP and UTP extended ECs to the pause and 10 μM GTP was added after 5 min to extend the RNA. For C370 delay assay (D), ECs were immobilized on Co2+ magnetic beads and extended to the pause by stepwise incubation with [α-32P] CTP, ATP, and CTP, incubated for 5 min, and then extended from the pause with CTP, ATP, and GTP (all at 10 μM NTP). ECs were washed five times with 1 ml EB between steps. (E, F) Denaturing RNA gels of products of assays depicted in (C) and (D). C, chase sample incubated with 1 mM all 4 NTPs. (G, H) C346 or C370 paused ECs formed as shown in (C) and (D) were resuspended in cleavage buffer (pH 9.0 and 20 mM Mg2+) to induce intrinsic cleavage (G) or in EB with or without 50 nM GreB to induce GreB-mediated hydrolysis (H). Possible 5′ and 3′ cleavage products determined by position of label are illustrated for C346 and C370 between the gel panels.