Volume 10, Issue 3, Pages 611-622 (September 2002) The σ70 Subunit of RNA Polymerase Is Contacted by the λQ Antiterminator during Early Elongation  Bryce E Nickels, Christine W Roberts, Haitao Sun, Jeffrey W Roberts, Ann Hochschild  Molecular Cell  Volume 10, Issue 3, Pages 611-622 (September 2002) DOI: 10.1016/S1097-2765(02)00648-2

Figure 1 λQ Function at PR′ (A) Presence of λQ allows RNAP that has initiated from PR′ to read through a transcription terminator. Blowups show functionally important elements at PR′ including the promoter −10 and −35 elements, the λQ binding element (QBE), and the pause-inducing −10-like element. Also indicated is a −35-like element positioned between the QBE and the pause-inducing −10-like element. (B) Sequence of events at PR′. Formation of the paused transcription complex at PR′ involves an interaction between σ70 region 2 and the pause-inducing −10-like element. λQ (shown as a dimer) then binds to the QBE and engages the paused transcription complex. Molecular Cell 2002 10, 611-622DOI: (10.1016/S1097-2765(02)00648-2)

Figure 2 λQ Interacts with Region 4 of σ70 (A) Fragments encompassing region 4 of σ70 interact with λQ by yeast two-hybrid analysis and affinity chromatography. The β-galactosidase activities reflect the interaction strengths; active segments are shown in red. The inset shows that σ70 fragment 526–613, encompassing region 4, binds λQ in an affinity chromatography assay: ft, flowthrough; w1, initial wash; w2, final wash; el, proteins eluted with imidazole. (B) Effect of λQ on transcription in vivo from pQBE-49. Cartoon shows design of promoter pQBE-49. Strain BN296 cells harboring the pQBE-49-lacZ fusion on an F′ episome and containing a plasmid encoding λQ (closed diamonds) or no λQ (open circles) were grown in the presence of the indicated concentrations of IPTG and assayed for β-galactosidase activity. (C) Effect of λQ on transcription from pQBE-61 in the presence of the α-σ70 chimera. Cartoon shows design of promoter pQBE-61. Assays were performed with cells harboring F′ episomes carrying the pQBE-61-lacZ fusion (strain BN297) or either of two derivatives, the pQBE-61/TTGACA-lacZ fusion (strain BN298) or the pQBE-61/TTAACA-lacZ fusion (strain BN299). The reporter strain cells contained plasmids pACλQtac and pBRα-σ70. Molecular Cell 2002 10, 611-622DOI: (10.1016/S1097-2765(02)00648-2)

Figure 3 Amino Acid Substitution A553D in Region 4 of σ70 Disrupts Interaction with λQ (A) Effects of substitution A553D in the σ moiety of the α-σ70 chimera on activator-dependent transcription from either pQBE-61/TTGACA (left panel) or placOR2-55/Cons-35 (right panel). β-galactosidase assays were performed with cells containing plasmids encoding the indicated proteins and grown in the presence of different concentrations of IPTG. (B) Effects of substitution A553D in chromosomally encoded σ70 on activator-dependent transcription from pQBE-49 (left panel) or λ promoter PRM (right panel). β-galactosidase assays were performed with cells encoding either wild-type σ70 or σ70 A553D and containing plasmids encoding the indicated activators. Molecular Cell 2002 10, 611-622DOI: (10.1016/S1097-2765(02)00648-2)

Figure 4 A553D Substitution in σ70 or Alteration of the TTGACT Motif Disrupts λQ-Mediated Antitermination In Vitro For each experiment, the percent of transcripts reading through the terminator (readthrough/readthrough plus terminated) in single-round assays is plotted. (A) Effects of the A553D substitution in σ70 on λQ-mediated antitermination in the presence or absence of NusA. Assays were performed with RNAP reconstituted with either wild-type σ70 or σ70 A553D (as indicated) in the presence of λQ (200 nM). (B) Effects of the G to A substitution in the TTGACT motif on λQ-mediated antitermination in the presence or absence of NusA. Assays were performed with wild-type RNAP on a PR′ template bearing either the wild-type or mutant TTGACT motif (as indicated) in the presence of λQ (400 nM). (C) Effects of the A553D substitution in σ70 on λQ-mediated antitermination in the presence or absence of the α-CTD, and in the presence of NusA. Assays were performed with RNAP reconstituted with either wild-type α (α WT) or a truncated derivative (α ΔCTD) and either wild-type σ70 or σ70 A553D in the presence of various concentrations of λQ. The percentage of readthrough transcription after 5 min incubation is plotted as a function of λQ concentration. (D) Effects of the A553D substitution in σ70 or the G to A substitution in the TTGACT motif on λQ-mediated antitermination in the absence or presence of a mutation (G-25C) in the QBE. Assays were performed with RNAP reconstituted with either wild-type σ70 or σ70 A553D on a PR′ template bearing either the wild-type or mutant TTGACT motif and either the wild-type or mutant QBE. The reactions contained NusA as well as λQ (500 nM). (E) Effects of the R588H substitution in σ70 on λQ-mediated antitermination. Assays were performed with RNAP reconstituted with either wild-type σ70 or σ70 R588H on a PR′ template bearing the G-25C mutation in the QBE and either the wild-type or mutant TTGACT motif. The reactions contained NusA as well as λQ (500 nM). Molecular Cell 2002 10, 611-622DOI: (10.1016/S1097-2765(02)00648-2)

Figure 5 A553D Substitution in σ70 or Alteration of the TTGACT Motif Disrupts λQ-Mediated Antitermination In Vivo (A) Schematic of PR′-lacZ fusion construct. Sequences extending from −109 to +232 of PR′ that include the natural terminator tR′ were fused to the lacZ gene. (B and C) Effects of the A553D substitution in chromosomally encoded σ70 or the G to A substitution in the TTGACT motif are revealed in the absence of NusA (B) or with a PR′ template bearing a weakened QBE (C). In (B), ΔnusA cells containing either wild-type σ70 or σ70 A553D and harboring a PR′-lacZ reporter with either the wild-type or mutant TTGACT motif were transformed with a plasmid that did or did not encode λQ as indicated. In (C), cells containing either wild-type σ70 or σ70 A553D and harboring a PR′-lacZ reporter with a mutation (G-25C) in the QBE and either the wild-type or mutant TTGACT motif were transformed with a plasmid encoding λQ. The cells were grown in the presence of no IPTG (B) or 5 μM IPTG (C) and assayed for β-galactosidase activity. In (C), the data are presented as percentages of the β-galactosidase activities measured in cells containing wild-type σ70 and the PR′ template with the wild-type TTGACT motif; the β-galactosidase values measured in the absence of λQ were roughly equivalent for all of the strains. Molecular Cell 2002 10, 611-622DOI: (10.1016/S1097-2765(02)00648-2)

Figure 6 A553D Substitution in σ70 or Alteration of the TTGACT Motif Destabilizes the Association of λQ with the Paused Elongation Complex In Vitro (A) Schematic of template used for exonuclease III challenge assay. PR′ DNA is end labeled at the 5′ end of the template strand as indicated (the red star). Positions at which the progress of exonuclease III digestion is blocked by bound protein are indicated: −32 (λQ-dependent barrier), −21 and −11 (σ70-dependent barriers), and −4 (core-dependent barrier). The red DNA segment is the pause-inducing sequence, shown bound to σ70 region 2 on the nontemplate strand. (B) Effect of the A553D substitution in σ70 on the stability of λQ's association with the paused elongation complex. Stalled elongation complexes were formed with RNAP reconstituted with either wild-type σ70 or σ70 A553D on a wild-type PR′ template. These complexes were incubated in either the presence or absence of 500 nM λQ and challenged with exonuclease III for the indicated times. (C) Effect of the G to A substitution in the TTGACT motif on the stability of λQ's association with the paused elongation complex. Stalled elongation complexes were formed with wild-type RNAP on a PR′ template bearing either the wild-type or mutant TTGACT motif. These complexes were incubated in either the presence or absence of 500 nM λQ and challenged with exonuclease III for 1 to 8 min as in (B). Molecular Cell 2002 10, 611-622DOI: (10.1016/S1097-2765(02)00648-2)

Figure 7 Model of the λQ-Engaged Paused Elongation Complex at PR′ The λQ-engaged paused elongation complex at PR′ is depicted. Regions 2 and 4 of σ70 are shown contacting the pause-inducing −10-like element and the −35-like element (TTGACT), which are separated by 1 bp. Molecular Cell 2002 10, 611-622DOI: (10.1016/S1097-2765(02)00648-2)