Strains and growth phenotypes. (A) Early log-phase cultures of strains with the rpoD and rpoC loci illustrated were serially diluted and plated onto LB plates or LB plates containing indole acrylic acid (IAA). Strains (top to bottom) are RL301, RL1374, CAG20153, and RL1094 (Supplementary Table 1). The efficiencies of plating minus IAA versus plus IAA were ∼10^-5 for the wild-type strain (CAG20153) and ∼1 for the β′;::σ⁷⁰ strain (RL1094). (B) Wild-type or β′;::σ⁷⁰ with TrpR-repressed rpoD strains (C600K^- and RL1094) were monitored during growth in LB at 37°C using a Klett colorimeter (data are averages for two independent cultures). After residing in stationary phase for ∼12 h, the strains were diluted back to Klett <5. (C) Model of σ⁷⁰ tethering to β′ based on the Thermus thermophilis holoenzyme structure (Vassylyev et al. 2002; β′ NCD removed). The upstream and downstream faces of RNAP are shown above schematics of the E. coli β′ and σ⁷⁰ subunits. RNAP subunits are shown in spacefill; σ, is shown as a C_α-trace colored by regions shown in the schematic. The N-terminal and C-terminal residues of σ and β′ resolved in the structure are depicted as black spheres (corresponding to β′1389 and σ⁷⁰91 in E. coli). σ region 1.1 (semitransparent white circle) is positioned based on the hydrated volume of an 82-amino acid globular protein attached to σ⁷⁰91.

Effective local concentration of tethered σ⁷⁰ measured by competitive binding. (A) Equilibrium σ⁷⁰-binding assay (see Materials and Methods). Samples are (left to right) 0.5, 1, 2, and 5 μM [³²P]σ⁷⁰ with 1 μM wild-type RNAP, β′;::σ⁷⁰ RNAP, or no RNAP. The positions of holoenzyme (E σ⁷⁰) and free σ⁷⁰ are indicated; slower σ⁷⁰ bands are σ⁷⁰ dimers. (B) Quantitation of A. The fraction of RNAP binding ³²P-labeled σ⁷⁰ is plotted against the amount of ³²P-labeled σ⁷⁰ present in the reactions. (•) Wild-type RNAP; (○) β′::σ⁷⁰ RNAP. Effective concentrations of σ⁷⁰ were estimated by nonlinear regression (Materials and Methods) from the averages of three experiments. (C) OC mobility assay. β′::σ⁷⁰ or wild-type RNAPs were incubated with the indicated amounts of σ⁷⁰ and then with added promoter DNA (Materials and Methods). OCs and DNA were separated on a native agarose gel and stained with EtBr. The positions of the free DNA, the wild-type OC, the β′::σ⁷⁰ OC, and the supershifted β′::σ⁷⁰ OC formed with a second, untethered σ⁷⁰ are indicated. Heparin was omitted in this experiment to minimize smearing of the supershifted OC band, although this caused variable nonspecific binding of free DNA (evident as aggregates near the top of the gel). Addition of heparin gave constant amounts of free DNA in gels and approximately the same amounts of supershifted OC, allowing use of up to 30 μM σ⁷⁰ (Fig. 4D). The slightly increased mobility of β′::σ⁷⁰ OCs (see also Fig. 2B) and of β′::σ⁷⁰ RNAP (panel A) may reflect a slight structural change caused by tethering that is under study. (β′::σ⁷⁰ RNAP does, however, contain ω.) (D) Quantitation of OC supershifting as a function of σ⁷⁰ concentration. The fraction of RNAP binding a second σ⁷⁰ (the supershifted species in C) is plotted against the concentration of added, untethered σ⁷⁰ (0.1, 0.5, 1, 10, and 30 μM; average of three experiments that included heparin). Predicted binding curves (assuming all σ⁷⁰ species to be equivalent for binding to RNAP) for different effective local concentrations of the tethered σ⁷⁰ are shown for comparison (Materials and Methods).

β′::σ⁷⁰ inhibits toxicity caused by σ⁷⁰RC584. RL113 (wild type, •), RL113 + pBADσ⁷⁰ (wt + ≥5× σ⁷⁰; ▴), or RL1366 (wt + β′::σ⁷⁰; ○) carrying pRM389 (σ⁷⁰RC584 expressed under LacI control) were plated with or without IPTG as described in Materials and Methods (both strains were deleted for lacY). Data are mean values of three to six independent experiments. The dotted line shows the approximate effect of wild-type σ⁷⁰ expressed from pCL391 for comparison.

The effective concentration of tethered σ⁷⁰ is sufficient to cause promoter-distal, σ⁷⁰-dependent pausing. The effective concentration of tethered σ⁷⁰ is sufficient to cause promoter-distal, σ⁷⁰-dependent pausing. Synchronous in vitro transcription with β′::σ⁷⁰ or wild-type RNAPs (Materials and Methods; time of transcript elongation as indicated in min; C, incubation with additional 200 μM NTP for 8 min after the last indicated time point). (A) +16/17 pause template (Ring et al. 1996). The top panel shows the run-off (RO) product; the lower panel shows the pause. The schematic of the template indicates the σ⁷⁰-binding sequence and the position of the pause. (B) +37 pause template (+20 in Ring et al. 1996). Gel panels and schematic are as in A. The three pause positions on this template have not been mapped precisely and therefore are designated 37a, 37b, and 37c; the third pause band (relative to the +16/17 site) is probably caused by the stronger consensus σ⁷⁰-binding sequence. The time dependence of pause RNA levels (plot) is the average of three independent experiments. (C) +462 pause template. Gel panels and schematic are as in A. σ (1 μM) or NusA (10 μM) were added as indicated.

Purified RNAP and immunoblot analysis. (A) (Left) Cellular extracts of wild-type (wt) or β′;::σ⁷⁰ (F) strains (RL301 and RL1094) were separated by 3%-8% Tris-Acetate (Novex), transferred to nitrocellulose, and probed with a mixture of β′ and σ⁷⁰ antibodies (Materials and Methods). Equal amounts of cellular protein were present in each lane. (Center) Western blot of wild-type (wt) and β′;::σ⁷⁰ (F) RNAPs. Samples were separated by 3%-8% Tris Acetate (Novex) and probed as in the left panel. (Right) β′;::σ⁷⁰ RNAP (F) purified from the strain containing the rpoC;::rpoD fusion and Trp-repressed rpoD (RL1094) separated by 4%-15% PAGE gel (Pharmacia) shown next to wild-type (wt) RNAP for size comparison. (B) β′;::σ⁷⁰ (F) or wild-type (wt) RNAPs were mixed with λP_R template, allowing the formation of open complexes at 37°C for 20 min (heparin was present where indicated for the last 10 min), separated by native gel electrophoresis (4% NuSieve agarose, 0.5× TBE), and stained with EtBr. The absence of heparin masked the slightly faster mobility of β′;::σ⁷⁰ OCs (see legend to Fig. 4C) because β′;::σ⁷⁰ OCs appeared to aggregate more than wild-type OCs.

(A) In vitro transcription assay. β′::σ⁷⁰ or wild-type RNAPs (40 nM) were allowed to initiate transcription on a linear DNA template (25 nM) with ApU, ATP, [³²P]CTP, and GTP. Samples were mixed with 2× STOP buffer at 10, 20, 30, 45, 60, 75, 90, and 120 sec. UTP was then added to allow transcription past A29, and additional samples were taken 10, 20, 30, 45, 60, 120, 240, and 480 sec later (final sample 10 min after reaction started). The samples were separated by 15% denaturing PAGE. (AUC) Trinucleotide abortive product; (A29) A29 EC; (P) his pause RNA; (RO) run-off RNA. (B) Kinetics of promoter association, following the mechanism defined by Saecker et al. (2002). k_f,obs and k_r,obs were obtained for β′::σ⁷⁰ and wild-type RNAPs on the template shown in panel A (≥3 independent experiments; Materials and Methods).

Heat-shock response. (A) Cells were grown in minimal media at 30°C and then shifted to 42°C. At the times indicated, samples were incubated with [³⁵S]methionine for 2 min. Samples were separated by 4%-12% PAGE. The positions of DnaK, GroE, and an 89-kD protein likely to be Lon are indicated. (^*) Major proteins whose expression decreases on heat shock; the top band is β′::σ⁷⁰, and the next-to-top band is β, β′. Relative GroE levels are averages of four experiments. (•) Wild-type (RL301); (○) β′::σ⁷⁰ strain with chromosomally encoded σ⁷⁰ (RL1374). (B) Immunoblot analysis using antibodies against σ³² (Materials and Methods). (Lanes 1-3) Wild-type whole-cell lysate (MC1060). (Lanes 4-6) β′::σ⁷⁰ whole-cell lysate (RL1454). (Lanes 7-9) His₆-tagged σ³². Equal amounts of total cellular protein were electrophoresed.

Model of σ⁷⁰-RNAP interactions during transcription in E. coli.