1. Volume 43, Issue 2, Pages 253-262 (July 2011)
The β Subunit Gate Loop Is Required for RNA Polymerase Modification by RfaH and NusG 
Anastasia Sevostyanova, Georgiy A. Belogurov, Rachel A. Mooney, Robert Landick, Irina Artsimovitch 
Molecular Cell 
Volume 43, Issue 2, Pages (July 2011)
DOI: /j.molcel
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2. Molecular Cell 2011 43, 253-262DOI: (10.1016/j.molcel.2011.05.026)
Copyright © 2011 Elsevier Inc. Terms and Conditions

3. Figure 1 RfaH Interactions with the EC
(A) AP-defective substitutions in RfaHN (Belogurov et al., 2010). The affected residues are shown as sticks and are colored according to the EC component with which these residues are proposed to interact in panel (B).
(B) A model of RfaHN bound to the EC (Belogurov et al., 2007). The T. thermophilus RNAP is shown as tubes with the bridge helix (β′BH) highlighted in cyan, the template DNA in black, the NT DNA in yellow, and the nascent RNA in red. The position of the RNAP active site is marked by the catalytic Mg2+ ion (a yellow sphere). RfaHN (blue) is bound to the NT strand and to β′CH (green); Tyr54 (forest green) interacts with the β′CH. The HTT cluster is positioned next to a mobile βGL (magenta). (A) and (B) were prepared with PyMol (DeLano Scientific).
(C) The RfaH retention assay on the linear pIA807 template is shown on top with the transcription start site (1) and end (224), the ops and the −10 elements, and the hisP signal indicated. Halted ECs were incubated with σ70 and the wild-type or an altered RfaH where indicated. Single-round transcription was restarted upon addition of NTPs and stopped at 10, 20, 40, 90, 180, and 360 s. In this and other experiments, some halted ECs become arrested and do not resume elongation. Positions of the G37, ops, σP, hisP, and run-off (RO) transcripts are indicated with arrows. The fraction of RNA at the σP site after a 360 s incubation (as % of total RNA) is presented below each panel; the errors (± SD) were calculated from four independent experiments.
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4. Figure 2 The βGL Deletion Eliminates AP Activity of RfaH
(A) Single-round pause assays on a pIA171 template that encodes the hisP signal, with or without 50 nM RfaHN. The half-lives of the wild-type, ΔβGL, and Δβ′SI3 RNAPs paused at the hisP site were determined as described in Landick et al., Samples were taken at 7, 15, 30, 45, 60, 80, 100, 120, 180, 300, and 540 s.
(B) Net elongation rates were determined on a “pause-free” pIA146 template as described previously (Svetlov et al., 2007) at 20 μM NTPs. RfaH was added to 80 nM where indicated. Samples were taken at 2, 3, 5, 7, 10, 20, and 40 min. In panels (A) and (B), the errors (± SD) were calculated from four independent experiments.
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5. Figure 3 RfaH Binds to the ΔβGL RNAP
(A) The σ competition assay was performed as in Figure 1C.
(B) The gel mobility shift assay was performed as in Artsimovitch and Landick, ECs assembled from DNA and RNA oligonucleotides and RNAP variants were incubated with [γ32P]ATP-labeled RfaH or [α32P]GTP (the incoming nucleotide specified by the template) and analyzed on an agarose gel.
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6. Figure 4 Polarity in the rfb Operon Is Controlled by RfaH and ρ
(A) The distribution of RNAP, RfaH, NusG, and ρ along the rfb operon revealed by the previously published ChIP-chip analyses (Belogurov et al., 2009; Mooney et al., 2009a).
(B) Polarity within the rfb operon evaluated by qRT-PCR. Total RNA was isolated from the wild-type or ΔrfaH cells grown in the presence or absence of 25 μg/ml BCM for 30 min and the amount of the RNA message in the rfbB region (red) was compared to that in the wbbI region (blue). The errors (± SD) were calculated from three independent experiments.
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7. Figure 5 Effects of the βGL Deletion on RfaH Function In Vivo
(A) Total RNA was isolated from cells expressing the wild-type or the ΔβGL RNAP in rfaH+ or ΔrfaH strain and the amount of wbbI RNA was compared to that in the rfbB region. See Figure S4 for the absolute levels of each message. The errors (± SD) were calculated from three independent experiments.
(B) Contributions of AP and NusG exclusion activities of RfaH to polarity. In lane 1, the RNAP is colored green to indicate the AP modification by RfaH. In lane 4, the fading NusG/ρ complex indicates its possible loss from the ΔβGL RNAP. Even though ρ may be bound to the RNAP throughout transcription and in the absence of NusG (Epshtein et al., 2010; Peters et al., 2009), here we omitted ρ from the RfaH+ lanes because our data show that ρ is largely inactive under these conditions.
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8. Figure 6
Interactions between GL and NusG Are Required for AP, but Not for ρ-Dependent Termination
(A) Pause assays on the linear pIA392 template shown on top with the scrambled ops (Artsimovitch and Landick, 2002) and the RO indicated. Halted wild-type A38 ECs were incubated with the wild-type or an altered NusG (at 100 nM) where indicated. Transcription was restarted upon addition of NTPs and rifapentin. Samples were taken at 5, 10, 20, 40, 90, 180, 300, and 600 s. Positions of the A38 and RO transcripts are indicated with arrows, the pause positions (at 39, 41, 43, and 45) are shown with a bracket. See Figure S5 for additional details. The fractions of the RO and scrambled ops RNAs are presented as % of total RNA on the right; the errors (± SD) were calculated from three independent experiments.
(B) Termination assays were performed on pIA267 template as described (Belogurov et al., 2009). Halted, [α32P]GMP-labeled ECs were formed at 40 nM with the wild-type or the ΔβGL RNAP. ρ and NusG were added to 10 and 100 nM, respectively, where indicated, followed by incubation with NTP substrates for 15 min at 37°C. NusG facilitates ρ-mediated RNA release, shifting the termination window upstream (marked by a bracket); the fractions of RNA in this region were determined from three independent experiments (± SD).
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9. Figure 7 Model of AP by RfaH
Two positions of the clamp domain are shown on a structural outline derived from the NTP-bound T. thermophilus EC (PDB ID 2o5j; Vassylyev et al., 2007) containing the helical hairpin form of the trigger loop (orange) packed against the bridge helix (cyan). The nucleic acid chains are omitted for clarity. The alternative trigger loop position observed in T. thermophilus holoenzyme (PDB ID 1i6h; Vassylyev et al., 2002) is also shown. An antitermination protein (AT, blue) can make bridging contacts between the GL (magenta) and clamp helices (green) across the DNA-binding channel. These contacts will prevent clamp opening observed in the T. aquaticus core RNAP (darker gray; PDB ID 1i6v; Zhang et al., 1999) to stabilize RNAP interactions with the DNA and increase its processivity.
Molecular Cell  , DOI: ( /j.molcel )
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