An Allosteric Path to Transcription Termination Vitaly Epshtein, Christopher J. Cardinale, Andrei E. Ruckenstein, Sergei Borukhov, Evgeny Nudler  Molecular Cell  Volume 28, Issue 6, Pages 991-1001 (December 2007) DOI: 10.1016/j.molcel.2007.10.011 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Termination Efficiency as a Function of Downstream DNA Duplex Strength (A) Alternative models of termination. The forward translocation model (right) postulates that the hairpin forces the EC to move forward without RNA synthesis. Such movement should lead to downstream DNA duplex unwinding and RNA extraction (Santangelo and Roberts, 2004). The allosteric model (left) postulates that the hairpin does not induce forward translocation but instead enters the RNA exit channel and main channel to melt the RNA:DNA hybrid and destabilize the EC (Gusarov and Nudler, 1999). Conformational change in the catalytic center (diamond) caused by the invading hairpin leads to irreversible EC inactivation. (B) Effect of downstream DNA changes on termination. Hairpin-proximal sequences of the tR2 terminator are indicated (top left). The red triangle shows the position of the catalytic center between the RNA 3′ terminus and the downstream DNA duplex. The green U indicates the termination site (U7 position). GC substitutions are shown in blue; AT substitutions are in pink. The radioautogram shows termination and full-length (runoff) RNA products from standard chase reactions (see the Experimental Procedures). The efficiency of termination (%T) is indicated under each lane. (C) Effect of downstream DNA changes on the termination rate of stalled ECU7. tR2 templates used are shown below. The orange G indicates the hybrid stabilizing substitution that prevents rapid hairpin folding and termination (Gusarov and Nudler, 1999). The initial immobilized EC was walked to the termination site at position +68 (U7) followed by washing with transcription buffer (TB) to remove unincorporated NTPs. After the time intervals indicated (min), a 200 μM mixture of CTP, ATP, and UTP (AT-rich template), or CTP, UTP, and GTP (GC-rich template) was added to extend the U7 transcript to +77 or +80, respectively. %T was calculated as a fraction of unchasable U7 transcripts. In the control reaction (W, wash lanes), inactive transcripts were washed away with TB after the chase, confirming their release during termination. Molecular Cell 2007 28, 991-1001DOI: (10.1016/j.molcel.2007.10.011) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Effect of the Front Element and RNAP Changes on the Termination Rate of the Stalled EC Modifications of the hairpin-proximal part of the tR2 terminator are colored. The orange G indicates the hybrid-stabilizing substitution that allows real-time measurement of the termination rate for each RNAP/modified terminator pair. AT-rich changes of the front element are pink; GC-rich changes are blue. The green U indicates the U7 termination site. The orange G indicates the hybrid-stabilizing substitution that prevents rapid hairpin folding and termination (Gusarov and Nudler, 1999). The red diamond between the hybrid and the front DNA duplex indicates the position of the catalytic center. For each modified terminator, the %T was determined as a fraction of unchasable and washable U7 transcript after 30 min of incubation. ΔT represents the change in %T compared to wild-type RNAP (WT) and the original front element (line 4). Clamp, β′1146–1148 mutant; zipper, β′50–52 mutant; G1136S, G loop fast mutant; I1134V, G loop slow mutant. Numbers represent the average values from three independent experiments with a standard deviation of less than 10%. Molecular Cell 2007 28, 991-1001DOI: (10.1016/j.molcel.2007.10.011) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Effect of RNAP Mutations on Termination (A) Effect of clamp mutations on termination. Bars in the left panel show the change of %T with respect to wild-type RNAP in a standard termination assay (Figure 1B). Numbers on the bars indicate triple Ala substitutions of corresponding amino acids in the β′ subunit. The right panel shows the effect of AT-rich and GC-rich downstream sequences on termination by the WT and β′1146–1148 mutant RNAP. %T was determined as the EC fraction terminated at the intact terminator in a standard chase reaction as described in Figure 1B. Error bars represent SD from three independent experiments. See Figure S8 for the location of mutants on the high-resolution structure of RNAP. (B) Effect of RNA exit channel mutations on termination. Colors represent mutant domains as indicated. Numbers on bars indicate triple Ala substitutions of corresponding amino acids in the β′ subunit. ΔZn indicates deletion of the entire zinc finger domain (β′ 35–107) (King et al., 2004). Δflap indicates substitution of the entire flap domain (β 884–917 aa) by a LeuValLeuGlu linker (Kuznedelov et al., 2002). Error bars represent SD from three independent experiments. Molecular Cell 2007 28, 991-1001DOI: (10.1016/j.molcel.2007.10.011) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Protein-RNA Interactions in the Active Center of the Termination Complex (A) Two models of termination result in different 3′-terminal RNA crosslinking. Semitransparent ovals represent the areas of 3′-terminal sU (green) crosslinking. In the case of the forward translocation model, crosslinking should occur 4 to 5 nt upstream of the catalytic site and target the nearby rifampicin-binding domain of the β subunit (evolutionarily conserved regions C or D) (Korzheva et al., 2000). In the allosteric model, the crosslinking should be restricted to the vicinity of the catalytic site made of the β′ subunit (evolutionarily conserved regions F, G, and D). (B) Twelve percent urea-PAGE shows [32P]RNA products from the derivatized ECsU7 before exposure to UV to induce crosslinking (UV) and after washing (W) with TB. RNA sequences of modified tR2 terminators are shown at the top. Green Us indicate crosslinkable 4-thio-UMP incorporated at the 3′ terminus in each case (see the Supplemental Data). Four percent SDS-PAGE shows protein-[32P]RNA crosslinked products. The major product corresponds to the β′ subunit in each case. The mobility difference between lanes 1, 3, and 2 is due to the covalently attached termination hairpin that is absent in the “no hairpin” sample. A control demonstrating specific crosslinking of RNA in the trapped complex as opposed to nonspecific crosslinking after potential posttermination rebinding to RNAP is presented in Figure S3. “−4 reference” lane represents the derivatized β subunit from a crosslinking molecule positioned 4 nt upstream of the catalytic center (Figure S4). (C) Summary of RNA crosslinking mapping results (see Figure S4 for mapping details). Horizontal bar represents the E. coli β′ subunit. Lettered boxes show evolutionarily conserved regions. DR stands for the dispensable region, which is a part of the G-helix-loop-helix (trigger-loop) domain. Location of crosslinking sites from each derivatized β′ probe is indicated by pink boxes. Molecular Cell 2007 28, 991-1001DOI: (10.1016/j.molcel.2007.10.011) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Role of G(Trigger)-Loop in Termination (A) Effect of G(trigger)-loop mutations on the termination rate of stalled ECU7. The hairpin-proximal sequence of the modified tR2 is indicated (top). Red triangle indicates position of the catalytic center between the RNA 3′ terminus and the downstream DNA duplex. Green U indicates the termination site (U7 position), and orange G indicates the hybrid-stabilizing substitution. The initial wild-type, fast (G1136S), and slow (I1134V) EC (Bar-Nahum et al., 2005) were immobilized on Co2+ beads and walked to the termination point followed by washing with TB to remove unincorporated NTPs. After the indicated time intervals (min), a 200 μM mixture of CTP, UTP, and GTP was added to extend the +68 (U7) transcript to +72. Percent (%) T was calculated as a fraction of unchasable U7 transcripts. In the control (W, wash lanes), the inactive transcripts were washed away with TB after the chase, confirming their release during termination. (B) Inhibition of termination by G(trigger)-loop targeting antibodies. ECU7 was prepared as in (A), followed by addition of anti-G loop 18B5 monoclonal antibodies (mAb; see the Experimental Procedures). In the control, the same amount of an anti-NusA mAb was used. After the indicated time intervals, aliquots were washed with TB (W lanes). Percent (%) T was calculated as a fraction of washed U7 transcripts. (C) Direct contact between the G(trigger)-loop and the hairpin loop during termination. The crosslinking probe (sU) was incorporated into the −15 position in the hairpin loop. The top panel (4% SDS-PAGE) shows protein-[32P]RNA crosslinking products (derivatized β′ and β subunits) from trapped (lanes 1 and 2), active (lanes 3 and 4), and binary (lane 5) complexes. To induce crosslinking, UV irradiation was performed in TB with the indicated amount of salt. Lane 5 was overexposed to compensate for the low intensity of radioactive material. The lower panel shows mapping of RNA crosslinking by single-hit cleavage at methionine (Met) and cysteine (Cys) residues with CNBr and NTCBA, respectively. Autoradiogram of gradient (6%–14%) SDS-PAGE shows products of partial degradation of the major crosslinked adduct in the trapped complex (marked by the red star in top and bottom panels). Schematics on the right show a summary of mapping results. Vertical bar represents the E. coli β′ subunit. Lettered boxes show evolutionarily conserved regions. DR stands for the dispensable region, which is a part of the G-helix-loop-helix (trigger-loop) domain. The red star indicates location of crosslinking. Molecular Cell 2007 28, 991-1001DOI: (10.1016/j.molcel.2007.10.011) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 Structural Model of Intrinsic Termination RNAP structures with explanatory schemes represent four possible stages of the termination process. Key structural elements are color coded: gray (green), nontemplate (template) strand of DNA; red, nascent RNA; yellow, β flap; violet, β′ lid; blue, β′ zipper; green, zinc finger; rose and gold, β lobes 1 and 2; aqua, β rudder; dark blue, G(trigger)-loop; and teal, DNA clamp and other parts of β′. Star, the catalytic center. (A) Paused EC. The first step involves a pause at the end of the U stretch, the role of which is to provide sufficient time for the hairpin to initiate folding (Gusarov and Nudler, 1999). (B) Hairpin nucleation. Nucleation of the hairpin could be initiated by interactions of the nascent RNA with the zinc finger and flap domains, which constitute the edge of the exit channel. The exit channel funnel may be wide enough to accommodate the hairpin head and a few base pairs of the stem, but to continue its growth the hairpin has to open the exit channel into a groove. (C) Hairpin incursion. The hairpin stem grows to 4 to 5 bp and begins invading the primary channel, unwinding the upstream end of the hybrid and initiating collapse of the upstream edge of the transcription bubble, while simultaneously pushing on the flexible β′ rudder and inducing opening movement of β lobe 2. This would result in widening of the main channel, which would allow the hairpin to move toward the downstream DNA and the secondary channel of RNAP. (D) Trapped complex. The hairpin stem grows to its final size of 7 to 8 bp while its head bends around the downstream edge of the transcription bubble, clashing with the G(trigger)-loop. This shortens the hybrid to about 3 bp, further collapsing the bubble. The clashing of the hairpin with the G(trigger)-loop may force it to move toward the active site and refold into a closed conformation, which would lock the enzyme in the pretranslocated state. Additionally, the hairpin action results in distortion of the hybrid, which could cause complete and irreversible enzyme inactivation. At the same time, G(trigger)-loop movement induces DNA-binding-clamp opening, resulting in release of the nascent RNA and disengagement of RNAP from the DNA template. (E) Different view of the trapped complex with G(trigger)-loop in unfolded conformation. The view is obtained by rotation of (D) around the vertical axis by ∼90° counterclockwise. The head of the hairpin is shown at interacting distance of ∼2 to 3 Å from the G(trigger)-loop. (F) Trapped complex with G(trigger)-loop refolded into a closed conformation and moved away from the hairpin by ∼5–7 Å. The view is shown in the same orientation as (E). Molecular Cell 2007 28, 991-1001DOI: (10.1016/j.molcel.2007.10.011) Copyright © 2007 Elsevier Inc. Terms and Conditions