Figure 6. Bridged H-NS filaments induced RNAP backtracking, which was rescued by GreB.

(A) Steps in pausing affected by the NusG-like N-terminal domain (NGN) of RfaH (RfaH-NTD) or Gre factors (e.g., GreB). Binding of the NGN RfaH-NTD (cyan) to the clamp domain (pink) of RNAP inhibits clamp motion and suppresses entry into pause states (Sevostyanova et al., 2011). The duration of pausing once paused ECs form can be increased by backtracking of DNA and RNA through RNAP, during which the 3′ RNA enters the RNAP secondary channel. GreB promotes endonucleolytic cleavage of the backtracked RNA in the RNAP active site to convert an offline paused EC back to an active EC (Laptenko et al., 2003). (B) Mean transcript lengths were averaged from two independent experiments. (C, D) Densitometry profiles of transcripts produced at 20°C, 12 mM Mg²⁺, and 1 mM each NTP from the λP_R-bgl template in bridged filaments (66 H-NS/kb) with or without 300 nM RfaH-NTD (C) or 50 nM GreB (D). Samples were removed at 0.33, 0.66, 1, 1.5, 2, 3, 4, 8, and 16 min after addition of NTPs and separated by denaturing PAGE.

DOI: http://dx.doi.org/10.7554/eLife.04970.017

Figure 6—figure supplement 1. At 1 mM NTPs, NusG partially suppressed H-NS effects on pausing and GreA more significantly suppressed H-NS effects.

(A) A26 ECs (10 nM) formed on λP_R-bgl were incubated with H-NS to form bridged filaments at 66 H-NS/kb and 12 mM Mg²⁺. GreA (500 nM) or NusG (75 nM) was added followed by NTPs (1 mM each), time points were taken at 0.5, 1, 1.5, 2, 4, and 8 min at 20°C, and separated by PAGE. M, labeled MspI-digested pBR322 marker. RO, run-off RNAs. (B) Mean transcript lengths were calculated from two independent experiments. (C, D) Densitometry profiles of transcripts produced from the λP_R-bgl templates with or without bridged H-NS and with or without GreA (C) or NusG (D).

DOI: http://dx.doi.org/10.7554/eLife.04970.018

Figure 6—figure supplement 2. Electrophoretic gel image showing that H-NS-induced RNAP backtracking was rescued by GreB.

PAGE corresponding to Figure 6, where reactions were assembled with 66 H-NS/kb and 50 nM GreB or 300 nM RfaH-NTD.

DOI: http://dx.doi.org/10.7554/eLife.04970.019

Figure 6—figure supplement 3. The H-NS stimulated C346 pause readily backtracked, whereas H-NS-resistant pausing at C370 occurred without obligate backtracking.

(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 EC_C346) or CTP, ATP, and GTP (for EC_C370) 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.

DOI: http://dx.doi.org/10.7554/eLife.04970.020