1. Structural Basis of Transcriptional Pausing in Bacteria
Albert Weixlbaumer, Katherine Leon, Robert Landick, Seth A. Darst 
Cell 
Volume 152, Issue 3, Pages (January 2013)
DOI: /j.cell
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3. Figure 1 Schematic of the Transcription Elongation Cycle
States for which X-ray crystal structures are available are highlighted in yellow. Pausing occurs as an off-pathway event with the formation of an initial elemental paused state, which further isomerizes into transcriptional pausing (backtracking or RNA hairpin) or termination (intrinsic or ρ-dependent) states. The elemental pause state crystallized in the present study is symbolized by the question mark.
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4. Figure 2 Tth RNAP Forms ePECs on a Minimal Nucleic Acid Scaffold
(A) (top) Schematic showing the scaffold variants used to assemble the elemental pause elongation complexes (ePECs) for pause kinetics and crystallization trials. The nontemplate DNA (ntDNA) is shown in blue; the template DNA (tDNA) in orange. Positions just before the target pause site (C15, blue), the target pause site (U16, red), and after pause escape (Esc, green) were compared. (bottom) Results from transcription kinetic assays: the inset shows the data points and model curves (using the branched kinetic scheme shown) for the C15, U16, and escaped transcripts (color-coded as in the scaffold schematic). The blow-up shows the U16 pause target site data. The red curve corresponds to the branched kinetic scheme containing the pause state; the dashed line is the best fit model that does not include the pause state. Typical for halted complexes, two rates of nucleotide addition were required to fit the C15 complexes (C15f and C15s). C15 complexes on the ePEC scaffold also exhibited pronounced pausing. Inclusion of an off-line pause state (U16p) was required to fit U16 on the PEC scaffold. Error bars depict standard deviations of six independent experiments.
(B) (top) Schematic showing the scaffold variants used to assemble the canonical elongation complexes (ECs) for kinetic analysis (color-coded as in (B); the same sequence was used in crystal structures of bacterial RNAP ECs; Vassylyev et al., 2007a, 2007b). (bottom) Results from transcription kinetic assays: the inset shows the data points and model curves (using the unbranched kinetic scheme shown) for the C15, G16, and escaped transcripts (color-coded as in the scaffold schematic). The blow-up shows the G16 data (corresponds to the U16 target pause site of the ePEC scaffolds). The red line is the best fit model that does not include the pause state. A simple one-state model with a 6-fold faster elongation rate than U16p (0.121 s−1 versus 0.017 s−1) accounted for all but a negligible fraction of G16 complexes on the EC scaffold. Error bars depict standard deviations of six independent experiments.
See also Figure S1 and Tables S1, S2, and S3.
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5. Figure 3 Overview of ePEC Structure
(A) An overview of the bacterial RNAP structure is shown as an α-carbon backbone ribbon, with color-coded subunits.
(B) Stereo image showing the unbiased difference Fourier map (contoured at 1.5σ) for the RNA/DNA hybrid in the Tth ePEC structure.
(C) The ePEC and EC (PDB ID code 2O5I; Vassylyev et al., 2007a) structures were aligned via their “core” modules (black). Differences in the orientation of the clamp domain are shown (ePEC clamp, green; EC clamp, yellow). Two orthogonal views are shown. In the ePEC, the clamp opens and loosens its grip on the nucleic acids (the RNA/DNA hybrid and downstream DNA duplex are shown as a molecular surface, color-coded as in the schematic of Figure 1B).
(D) Differential nucleic acid solvent accessibility (with versus without the RNAP) for the EC (left) and ePEC (right). At the top, the same view of the nucleic acid scaffold (color-coded as in Figure 1) is shown, based on a structural superposition of the EC and ePEC core modules. At the bottom, the molecular surface of the scaffold is colored by relative loss of solvent accessibility caused by the interactions with RNAP (blue, no loss; red, 100% loss). In an ePEC (right), the tDNA in particular is more solvent exposed, indicated by a reduced loss of solvent accessibility upon binding to RNAP (blue).
See also Figure S2.
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6. Figure 4
Comparison of RNAP Active Site Region for ePEC and Canonical EC Structures
(A) An omit map, (blue mesh, contoured at 1.3σ) confirms the kinked BH in the ePEC (green) compared to an EC (gray).
(B) View of the ePEC kinked BH (green), along with the tDNA (orange) and RNA transcript (black) near the active site. A backbone trace of Sw2 is also shown (green). Superimposed are the base-paired +1 tDNA and nucleotide substrate from the EC (outlined as red dashed lines; Vassylyev et al., 2007b), as well as the EC position of Sw2 (light gray). The kinked ePEC BH sterically clashes with the superimposed +1 tDNA and less severely with the nucleotide substrate. As a consequence, the +1 tDNA base sits in an alternate position at the side of the BH.
(C) View of the relatively straight BH of the EC, which leaves room for the +1 tDNA and incoming nucleotide substrate (yellow). Sw2 is also shown (yellow).
See also Figure S3.
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7. Figure 5
Comparison of Key Nucleic Acid Contacts for ePEC and Canonical EC Structures
(A) Schematic representation of RNAP contacts to the nucleic acids in an EC (PDB ID code 2O5I; Vassylyev et al., 2007a). Text color corresponds to the RNAP protein element. Colored boxes indicate if a contact is maintained (gray) or lost/suboptimal (red) upon entering the elemental paused state.
(B) Same as (A) but for the ePEC. Newly formed contacts in the ePEC are highlighted by green boxes.
(C) Changes in contacts to the nucleic acids upon entering the elemental pause are exemplified by conformational changes in Sw1–3 and the lid (directions of movement from the EC to the ePEC structure are indicated by arrows).
See also Figure S4.
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8. Figure 6
Comparison of the RNA Exit Channel in the ePEC and Canonical EC Structures
(A and B) In the ePEC (A), the RNA exit channel is wider compared to the EC (B). An RNA pause hairpin was modeled in the RNA exit channel. Although it can be accommodated in the ePEC without major steric clashes, the exit channel seems to be too narrow in the EC.
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9. Figure 7
Schematic Representation of Our Model for Transcriptional Pausing
During translocation, RNAP has an opportunity to relax to the open-clamp conformation if it fails to reform polar contacts to the nucleic acids, thus entering the elemental paused state. Key features of the ePEC are an open clamp, the kinked BH (red circle) blocking tDNA access to the active site, and a widened hybrid binding site and RNA exit channel. From the ePEC: (1) the RNAP can backtrack (backtrack pause), (2) an RNA pause hairpin can form, wedging open the clamp in the pretranslocated hairpin pause, or (3) a terminator hairpin can form, resulting in dissociation of the RNAP from the nucleic acids (termination).
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10. Figure S1
Tth RNAP Responds to the Eco his Pause Signal, Related to Figure 2
(A) Sample gels showing raw data for minimal scaffold pause assays. Time course to compare pause inducing minimal scaffolds lacking a hairpin with the scaffold used for the canonical EC halted one base prior (C15) to the target site (U16/G16). Scaffold 2_1_10 is shown (top gel; compare Table S1 and Figure 2) along with the EC scaffold (EC1; bottom gel; compare Table S1 and Figure 2). Relevant positions (C15, U16/G16, and Esc) are indicated using the same color code as for graphs in Figure 2. Samples were taken after addition of the next two nucleotides following position 15. Results for scaffold 2_7_10 (compare Table S1) are essentially identical (data not shown).
(B) Tth RNAP RNA-hairpin PECs respond to NusA. Comparison of pause escape kinetics for Tth PECs halted at the pause site (U29) using a minimal scaffold (2_1_1; compare Table S1) containing an RNA hairpin in absence and presence of Tth NusA (top and bottom gel respectively). Samples were taken after addition of GTP. NusA (compare blue versus green curve) has a 5-10-fold effect on the pause escape rate and pause half-life. Results for Taq RNAP using the same scaffold are essentially identical (data not shown). Error bars depict standard deviations of 3 independent experiments.
(C) Tth RNAP transcription elongation assays. Comparison of promoter initiated transcription assays in absence and presence of NusA. The wt Eco his pause sequence (hisP), the his pause sequence lacking the hairpin (hisP lacking hairpin), or the sequence used to crystallize a canonical EC (PDB ID 2O5I EC sequence) were embedded in a longer transcription unit under the control of the T7A1 promoter. Halted ECs (A29) were formed by omission of one NTP. Transcription was restarted by addition of all 4 NTPs and samples were taken at predetermined time points. Pausing of Tth RNAP was observed for the wt his pause as well as for the his pause lacking the hairpin. In the presence of NusA, pausing was more pronounced (red arrows, top 2 gels, gray box indicates relevant sequence as determined by 3′-deoxy-A sequencing). In contrast, the sequence used for the canonical EC did not show any pause at the relevant position (green arrow, bottom gel). Only relevant parts of gels are shown.
(D) Eco RNAP transcription elongation assays. As a control, the assays described in Figure S1C were repeated with Eco RNAP. A strong pause was observed for the wt his pause as well as the his pause lacking the hairpin at the same position as for Tth RNAP (red arrow). Both pauses were more pronounced in presence of NusA. Only the position of the his pause is shown on the gel.
(E) Transcription elongation assay template sequences. Alignment of the non-template DNA sequence used in Figures S1C and S1D. The position of halted ECs (A29) as well as the relevant embedded sequences and target sites (gray box, red and green arrows; compare to Figure S1C) are indicated.
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11. Figure S2
Electron Density Maps of ePEC Crystal Structures and Comparison with Other RNAP Structures, Related to Figure 3
(A) RNA/DNA hybrids. Stereo image showing the unbiased positive difference Fourier map for the RNA/DNA hybrid in the R3 crystal form (contoured at 2σ) of the Tth ePEC structure as well as for a low resolution data set from the Taq ePEC structure (contoured at 1σ).
(B) Downstream duplex DNA and β′NCD. Stereo image for unbiased positive difference Fourier maps of the downstream duplex in the P3121 crystal form of the Tth ePEC structure calculated to 5 Å resolution (left; contoured at 1.4σ). After molecular replacement and one round of rigid body refinement using the canonical EC (PDB ID 2O5I) unbiased positive difference Fourier density clearly indicated conformational changes (the non-conserved domain forming the tip of the β′ pincer is shown; contoured at 1σ).
(C) Clamp conformational change for Taq ePEC structure. Despite the low resolution for the Taq ePEC structure, a molecular replacement solution was obtained using a Taq EC model. An ePEC model with an open clamp fit the resulting 3mfo-2Dfc electron density maps better than the EC model, confirming that the clamp is also in the open position in the Taq P41 crystal form.
(D) Comparison of ePEC structures with other RNAP structures. Superposition of the 3 crystal forms obtained for ePECs (Tth P3121, Tth R3, and Taq P41), with Taq RNAP core (PDB ID 1HQM) and a canonical EC (PDB ID 2O5I). In the EC, the clamp is closed (yellow), but in the ePECs as well as in the structure of RNAP core, the clamp is open (green, blue, purple, and salmon respectively, compare Table S4).
(E) Comparison of crystal packing in the three ePEC crystal forms. Crystal contacts (red spheres) of symmetry related molecules (gray) with the clamp (orange) and β′ NCD (blue) are shown for the three crystal forms (Tth P3121 left; Tth R3 middle; Taq P41 right). While some of the contacts between the P3121 and R3 crystal form are the same, most of these are made to the β′ NCD, which is very flexible (and has in fact a different orientation in the two crystal forms). The packing in the Taq P41 crystal form is entirely different. As a consequence, opening of the clamp is unlikely an artifact of crystal packing.
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12. Figure S3
E Site and +1 Template DNA Position in ePEC, Related to Figure 4
(A) Position of the +1 template DNA base. Unbiased positive difference Fourier density at low contour level suggests that the template DNA base at the +1 position flips out with its Watson-Crick edge facing the bridge helix.
(B) Comparison of active site conformation between an EC and the ePEC. Soaking of a non-hydrolyzable nucleotide analog resulted in strong positive difference Fourier density in the E-site, a nucleotide binding site identified previously (green mesh, contoured at 3σ) (Westover et al., 2004). This is consistent with the absent +1 template DNA base in the active site. In addition, modeling suggests a steric clash between the kinked bridge helix (green) of the ePEC and the substrate position in an EC (yellow).
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13. Figure S4
The Upstream End of the RNA/DNA Hybrid Is Different in the ePEC Compared to an EC, Related to Figure 5
(A) Cross crystal averaging confirms RNA/DNA hybrid conformation in ePEC. The electron density maps resulting from cross-crystal averaging as implemented in Phenix confirm the overall RNA/DNA hybrid conformation. In agreement with unbiased difference Fourier maps, the electron density for the DNA is consistently weaker (compare contouring levels for RNA and DNA) in the available ePEC structures. This is consistent with the loss in polar contacts between RNAP modules and the template DNA but not the RNA.
(B) Comparison of RNA/DNA hybrid between EC and ePEC. While the downstream end of the RNA/DNA hybrid is virtually identical between EC and ePEC, the upstream end is different with the base pairs at −8 and −9 being more tilted relative to the helix axis.
(C) Comparison of upstream RNA/DNA hybrid from ePEC versus EC. While the downstream part of the RNA/DNA hybrid is virtually identical between the ePEC (RNA black) and a canonical EC (RNA gray), the upstream end of the RNA seems to get pulled in the direction of exit as reflected by the difference in phosphate positions. As a result, the upstream base pairs at −8 and −9 are more tilted and seem to deviate from Watson-Crick geometry. Unbiased positive difference Fourier density as well as simulated annealing omit maps (SA-omit maps) from cns DEN refinement confirm the RNA backbone conformation. The SA-omit map was calculated with phases averaged from 20 models, which were the result of cns DEN refinement using a starting model that has never been refined against an ePEC nucleic acid scaffold.
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