Isolation and Characterization of σ70-Retaining Transcription Elongation Complexes from Escherichia coli  Gil Bar-Nahum, Evgeny Nudler  Cell  Volume 106, Issue 4, Pages 443-451 (August 2001) DOI: 10.1016/S0092-8674(01)00461-5

Figure 1 Quantification of the σ70 Release from EC (A) EC purification scheme. EC32 was formed in solution followed by annealing of the complementary 3′-biotinylated DNA oligo to its nascent transcript. Avidin beads were added to the reaction followed by washing with TB and ribonucelase A (RNase) treatment. Supernatant was loaded onto 4%–12% SDS PAGE. The gel was transferred onto nitrocellulose and antibodies against α, β, and σ were employed. (B) RNA products from the transcription reaction with EC32 prepared as in (A). The washed EC32 in TB (lane 1) was walked one step to the position +33 (lane 2) or chased with 1 mM NTPs (lane 3). tR2 stands for the termination product. (C) Western blotting. The panel shows antibody stained RNAP subunits. Samples were prepared using the holoenzyme isolated from the E. coli culture harvested at stationary phase (S) (lanes 1–3 and 7–9) or exponential phase (E) (lanes 4–6). Lanes 2, 3, 5, 6, 7, and 8 show the supernatant fractions of RNase-treated EC32. EC32 was prepared with or without the biotinylated oligo (B-oligo). Without B-oligo, EC32 could not be recovered as evident from corresponding “empty” lanes. This control excludes the possibility of nonspecific binding to the beads. Where indicated, RNAP core (2 pmol) and DNA (10 pmol) were added to EC32 for 10 min prior to the addition of B-oligo. The procedure shown in (A) was also performed in the absence of ribonucleotides (−NTP) (lane 10) or with pure σ70 instead of the holoenzyme (lane 11). Percentage of σ70 in EC32 (lanes 2, 5, and 8) compared to pure input holoenzymes (Holo) is indicated at the bottom (lanes 1, 4, and 7). (D) Summary data for σ70 release obtained from three Western blotting experiments, conducted with six independent holoenzyme preparations (three from stationary phase and three from the exponential phase) Cell 2001 106, 443-451DOI: (10.1016/S0092-8674(01)00461-5)

Figure 2 σ70-RNA Contacts in Elongation (A) Protein-RNA cross-linking in EC. Schematic on the top depicts EC20 carrying [32P]RNA, which is immobilized on Ni2+ beads via his-tagged β′ subunit. Cross-linkable 5 - ( p - azidotetrafluoro ) - benzamino - allil - UTP (AzU) or 4-thio-UTP (sU) were incorporated at position +21 and EC was walked to +30, +38, +43, +50, +54, and +61 such that AzU appeared 9, 17, 22, 29, 33, and 41 nt upstream of the RNA 3′ terminus. At each position, cross-linking was photo-activated with UV light. Autoradiograms show protein-RNA cross-linking products from each EC walking step (+30, +38, and +43 [left panel], and +43, +50, +54, and +61 [right panel]). “−nt” indicates the distance of cross-linker from the 3′ end of nascent RNA. RNA products from upper right panel are resolved on high percentage urea PAGE (lower panel). CpApUpC serves as a marker for shorter RNA products. (B) Mapping of RNA cross-links by single-hit cleavage at Met residues. The autoradiogram of a gradient (7% to 14%; 20 cm) SDS-PAGE shows products of 5 min partial degradation of the “σ70” species from the upper left panel with CNBr. Numbers indicate Met positions in σ70. As a reference marker, CNBr degradation products of the β subunit labeled near Met1234 are shown in lane 4. Numbers on the right and left indicate the size of the fragment in amino acids (aa) Cell 2001 106, 443-451DOI: (10.1016/S0092-8674(01)00461-5)

Figure 3 Isolation and Characterization of EC-σ70 (A) Autoradiogram demonstrates [32P]RNA products isolated from EC20 formed with different RNAP holoenzymes. Lanes 1 and 2 represent EC20 formed with the non-his-tagged holoenzyme before or after it was applied to Ni2+ beads and washed with TB. Lanes 3 and 4 represent EC20 formed with the C-terminal his-tagged β′ holoenzyme (β′his) before or after it was applied to Ni2+ beads and washed with TB. The rest of the autoradiogram shows EC20 formed with the N-terminal his-tagged σ70 holoenzyme (σ70his) (lanes 5–10). Lanes 5, 9, and 10 represent EC20-σ70his before or after it was applied to Ni2+ beads and washed with TB or 200 mM KCl. A1 promoter DNA (10 pmol, lane 6), NusA (100 nM, lane 7), or RNAP core (2 pmol, lane 8) were added prior to EC20-σ70his was immobilized onto Ni2+ beads and washed with TB. The relative recovery of RNA in % is shown at the bottom. (B) Walking with immobilized EC-σ70. The initial stalled EC-σ70his was prepared at three different templates containing the gal P1 promoter (left panel), rrnB1 P1 (center panel), and T7A1 (right panel). Each template has the tR2 terminator near the 3′ end. Autoradiograms show [32P]RNA isolated from the immobilized EC-σ70his that was walked four steps in TB to the positions indicated at the bottom of each panel (lanes 1–4, 7–10, or 13–16) or chased with all four NTPs in the absence (lanes 5, 11, and 17) or presence (lanes 6, 12, and 18) of 30 nM NusA. “tR2” stands for termination products Cell 2001 106, 443-451DOI: (10.1016/S0092-8674(01)00461-5)

Figure 4 RNA Trajectory in EC and EC-σ70 (A) Schematic of three T7A1 promoter templates (a, b, and c) with varying distances between the EcoRI restriction endonuclease site and the start of transcription. (B) Experimental design. The mutant EcoRI protein, EcoRQ111, was used as a roadblock to stall EC at three different positions determined by each template. The initial EC20 carrying [32P]RNA was formed with his-tagged σ70 or his-tagged β′ holoenzymes and chased to the roadblock in TB in the presence 150 μM ATP, 150 μM CTP, 150 μM GTP, and 20 μM AzUTP. After induction of cross-linking with UV, each reaction was split with one fraction remaining in solution while the other was applied to Ni2+ beads to purify EC-σ70. The washed immobilized fractions and solution fractions were then denatured and loaded on SDS PAGE (4%–12%). (C) The autoradiogram of β, β′, σ70, and α subunits labeled by RNA cross-linking. “Solution” stands for the total populations of EC in the solution fractions (lanes 1–6) and “Ni-beads” stands for the washed immobilized fractions (lanes 7–12). “σ70his” stands for the holoenzyme carrying his-tagged σ70 and “β′his” stands for the holoenzyme carrying his-tagged β′ Cell 2001 106, 443-451DOI: (10.1016/S0092-8674(01)00461-5)

Figure 5 Multiround Transcription by EC-σ70 (A) The assay. The initial ECs were formed with the his-tagged σ70 holoenzyme on templates with three different promoters (T7A1, gal P1, rrnB P1), but identical transcribed regions. In each case, one fraction was applied to Ni2+ beads (for purification of EC-σ70) and the other remained in solution (the total population). Immobilized EC-σ70his was washed with TB, released from beads with imidazole, and diluted in TB. Multiple rounds of transcription were initiated in the EC-σ70 fraction and total population by adding the 10× “multiround mix” (150 μM CpApUpC, 10 mM ATP, 7.5 mM GTP, 1.75 mM UTP, 300 μM CTP, 1 μM [32P]CTP, 2 pmol DNA, and RNase inhibitor) and 30 nM NusA. Aliquots were taken at 5 min intervals. Where indicated, rifampicin (Rif, 5 μg/ml) was added prior to the multiround mix to limit the reaction to a single round (single). (B) Autoradiograms show RNA termination products of the total EC population (upper panels) and EC-σ70 (lower panels). Radioactive bands were quantitated and normalized to the 20 min single round (Rif) product in each respective experiment and relative intensities within each pair were plotted. (C) Bifurcation of the transcription cycle. Two populations of RNA polymerases (R) are involved in transcription in E. coli, one that releases σ70 in elongation (σ:R) and one which does not (σ:R'). Pc corresponds to the closed promoter complex, Po—the open complex, and kf—the rate constant of the reaction. Promoters for which kσRf is rate limiting (e.g., A1) would have a higher rate of initiation with σ:R′ than σ:R due to faster recycling Cell 2001 106, 443-451DOI: (10.1016/S0092-8674(01)00461-5)