A Transcription Antiterminator Constructs a NusA-Dependent Shield to the Emerging Transcript  Smita Shankar, Asma Hatoum, Jeffrey W. Roberts  Molecular Cell  Volume 27, Issue 6, Pages 914-927 (September 2007) DOI: 10.1016/j.molcel.2007.07.025 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Construction of Stalled Q82-Modified or Unmodified Complexes (A) Schematic of the experimental method used to construct defined populations of Q82-modified complexes. The template was obtained from pSS100 and contained the phage 82 late gene promoter, the intrinsic terminator t82, a C-rich region for optimal Rho binding to the RNA, and an EcoR1 site. Distances are indicated from the transcription start site. (B) Gel resolution of elongation complexes filtered past t82 using either the oligo anti-t82 or the antiterminator Q82, and stalled at a downstream site using the roadblock protein EcoR1-Gln111. After incubation with the indicated components, reaction mixtures were separated into magnetic bead-bound pellet (P) and supernatant (S) fractions. Numbers in parentheses indicate lengths of the RNA transcripts. Molecular Cell 2007 27, 914-927DOI: (10.1016/j.molcel.2007.07.025) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Stalled Elongation Complexes as Models for Transcription Termination Mechanisms and Effect of NusA on Q82-Mediated Stabilization of Stalled Elongation Complexes on a Template from pSS100 (A) Stalled unmodified complexes, made and maintained in the presence of NusA, were incubated at 37°C for 10 min with oligos that annealed at different distances from the RNA 3′ end, as shown in the schematic (the stalled complexes may include some backtracked complexes not shown in the schematic), and transcript release efficiency was measured. Quantification of the data in the gel (left) is shown in the graph (right). (B) Stalled Q82-modified or unmodified complexes, made and maintained in the presence or absence of NusA, were incubated at 37°C for 10 min with a set of oligos of varying lengths, 1 μM each, all annealing at their 5′ end to −9 of the transcript. (C) Stalled Q82-modified or unmodified complexes, made and maintained in the presence or absence of NusA, were incubated at 37°C for 10 min with varying concentrations of the 15-mer oligo that extends from −9 to −23 of the transcript. (D and E) Time course of RNA release from stalled Q82-modified or unmodified complexes, made and maintained in the presence or absence of NusA, and treated with (D) 1 μM 15-mer oligo or (E) 10 nM Rho and 1 mM dATP. Molecular Cell 2007 27, 914-927DOI: (10.1016/j.molcel.2007.07.025) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Effect of NusA on Q82-Mediated Antitermination In Vitro (A) Template p82a containing the wild-type phage 82 late gene promoter and the t82 intrinsic terminator segment was transcribed in the absence or presence of NusA, with 0, 3.1, 6.2, 12.5, 25, 50, and 100 nM Q82. The percent readthrough was calculated as the moles readthrough RNA (RT) divided by the sum of moles RT and moles terminated RNA (T), minus background. (B) Template pXY312 containing wild-type phage 82 late gene promoter upstream of the λ tR1 Rho-dependent termination sites was transcribed as in (A), except in the presence of 54 nM Rho, with 0, 6.2, 12.5, 25, 50, 100, and 200 nM Q82. (C) Complexes were synthesized to +29 by CTP deprivation on template pAH100 in the presence or absence of NusA and Q82 as described. Transcription buffer was replaced and synthesis was continued in the presence or absence of NusA and Q82 as indicated, with 100 μM CTP, ATP, and GTP, and 1, 2, 5, 10, 20, or 100 μM UTP. Molecular Cell 2007 27, 914-927DOI: (10.1016/j.molcel.2007.07.025) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Effect of Q82 on the Stability of Elongation Complexes against the Three Types of Termination A time course of RNA release from stalled Q82-modified or unmodified complexes, made and maintained in the presence of NusA on a template from pSS100, is shown, in the presence of 1 μM 15-mer release oligo (A); 10 nM Rho + 1 mM dATP as energy source (B); and 50 nM Mfd + 1 mM dATP as energy source (C). Molecular Cell 2007 27, 914-927DOI: (10.1016/j.molcel.2007.07.025) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Effect of Q82 on RNA Accessibility Probed with Oligos and RNase H Stalled Q82-modified or unmodified complexes were used, made and maintained in the presence of NusA on a template from pSS100. (A) Schematic of the set of nested oligos that were used as probes for RNA accessibility studies with RNase H. (B) A time course of digestion is shown using the oligo that anneals to −14 of the transcript. (C) Effect of oligo position on RNA protection. Complexes were incubated for 10 min at 37°C with 1 μM each oligo in the presence of RNase H as described. Molecular Cell 2007 27, 914-927DOI: (10.1016/j.molcel.2007.07.025) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 Q82-Mediated Protection of RNA Emerging from a Transcription Elongation Complex (A) RNase A footprint time course of RNA labeled at the 3′ penultimate position in Q82-modified and unmodified stalled complexes made in the presence of NusA on a template from pSS100. (B) RNase I footprint time course of 5′ end-labeled RNA in Q82-modified and unmodified complexes made in the presence of NusA, stalled artificially at position 25 in the absence of CTP, and then advanced to position 76 in the absence of UTP, on a template from pSS418. Molecular Cell 2007 27, 914-927DOI: (10.1016/j.molcel.2007.07.025) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 7 Role of NusA in Q82-Mediated RNA Protection against RNase H Digestion (A) Q82-modified and unmodified complexes were elongated to the EcoR1 stall site of the template pSS100 in the absence or presence of NusA, and NusA was either excluded from or included in the resuspension buffer. The complexes were treated with RNase H in the absence or presence of the oligo probe that anneals to −14 of the transcript. (B) Q82-modified and unmodified complexes, containing either wild-type RNAP holoenzyme or RNAP-αΔCTD, were elongated to the EcoR1 stall site of the template pSS100 in the presence of NusA. The complexes were washed in the presence of NusA and treated with RNase H in the absence or presence of the oligonucleotide probe that anneals to −14 of the transcript. (C) A model suggesting a location of NusA and Q82 near the RNAP β flap and the RNAP-αCTD, all the protein components interacting directly or indirectly with the transcript. Molecular Cell 2007 27, 914-927DOI: (10.1016/j.molcel.2007.07.025) Copyright © 2007 Elsevier Inc. Terms and Conditions