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Comparative Study
. 2003 Nov 15;17(22):2839-51.
doi: 10.1101/gad.1142203.

Tethering sigma70 to RNA polymerase reveals high in vivo activity of sigma factors and sigma70-dependent pausing at promoter-distal locations

Rachel Anne Mooney  1 , Robert Landick
Affiliations
Comparative Study

Tethering sigma70 to RNA polymerase reveals high in vivo activity of sigma factors and sigma70-dependent pausing at promoter-distal locations

Rachel Anne Mooney et al. Genes Dev. .

Abstract

Bacterial sigma factors compete for binding to RNA polymerase (RNAP) to control promoter selection, and in some cases interact with RNAP to regulate at least the early stages of transcript elongation. However, the effective concentration of sigmas in vivo, and the extent to which sigma can regulate transcript elongation generally, are unknown. We report that tethering sigma70 to all RNAP molecules via genetic fusion of rpoD to rpoC (encoding sigma70 and RNAP's beta' subunit, respectively) yields viable Escherichia coli strains in which alternative sigma-factor function is not impaired. beta'::sigma70 RNAP transcribed DNA normally in vitro, but allowed sigma70-dependent pausing at extended -10-like sequences anywhere in a transcriptional unit. Based on measurement of the effective concentration of tethered sigma70, we conclude that the effective concentration of sigma70 in E. coli (i.e., its thermodynamic activity) is close to its bulk concentration. At this level, sigma70 would be a bona fide elongation factor able to direct transcriptional pausing even after its release from RNAP during promoter escape.

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Figures

Figure 1.
Figure 1.
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 Cα-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.
Figure 2.
Figure 2.
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 λPR 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.
Figure 4.
Figure 4.
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).
Figure 3.
Figure 3.
(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). kf,obs and kr,obs were obtained for β′::σ70 and wild-type RNAPs on the template shown in panel A (≥3 independent experiments; Materials and Methods).
Figure 5.
Figure 5.
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.
Figure 6.
Figure 6.
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.
Figure 7.
Figure 7.
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.
Figure 8.
Figure 8.
Model of σ70-RNAP interactions during transcription in E. coli.
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