Crystal Structure of a Transcribing RNA Polymerase II Complex Reveals a Complete Transcription Bubble

Abstract

Notwithstanding numerous published structures of RNA Polymerase II (Pol II), structural details of Pol II engaging a complete nucleic acid scaffold have been lacking. Here, we report the structures of TFIIF-stabilized transcribing Pol II complexes, revealing the upstream duplex and full transcription bubble. The upstream duplex lies over a wedge-shaped loop from Rpb2 that engages its minor groove, providing part of the structural framework for DNA tracking during elongation. At the upstream transcription bubble fork, rudder and fork loop 1 residues spatially coordinate strand annealing and the nascent RNA transcript. At the downstream fork, a network of Pol II interactions with the non-template strand forms a rigid domain with the trigger loop (TL), allowing visualization of its open state. Overall, our observations suggest that "open/closed" conformational transitions of the TL may be linked to interactions with the non-template strand, possibly in a synchronized ratcheting manner conducive to polymerase translocation.

Figure 1. Architecture of the complete nucleic acid scaffold

A. Difference F_obs-F_calc electron density map contoured at 2σ. The following color scheme will be used throughout: cyan, template strand; green, non-template strand; red, RNA transcript. B. Front and side views of the 2F_obs-F_calc map for the final refined map contoured at 1.0σ. C. Cartoon representation of the 38-nucleotide refined nucleic acid scaffold; downstream and upstream duplexes form an angle of approximately 130° degrees. D. Surface representation of the overlay between Δ4/7-TC (blue) and Pol II-TC (grey). The two structures overlay remarkably well, minor structural differences occur at the downstream fork (see also Fig. S1).

Figure 2. Overall structure of Δ4,7-TC

A. Surface representation (side view, Rpb2 removed) illustrating the position of the scaffold inside Pol II. Wedge, jaw and arch interactions with the scaffold lie on almost a perfect plane, possibly to minimize strain during elongation. B,C. The scaffold binds asymmetrically inside Pol II’s cleft, more prominently at the downstream end where observed clamp-DNA distances of ≈6 Å vs. lobe DNA distances of ≈14 Å are due to interactions with clamp and jaw residues (see also Fig. S2). D. Surface electrostatic representation calculated using the APBS (Baker et al., 2001) suite in PyMOL to illustrate how the non-template strand follows a path of positively charged residues inside Pol II’s cleft (lobe and protrusion). The final refined 2F_obs-F_calc map (grey) contoured at 1.0σ is also illustrated to show the continuous density for the DNA scaffold.

Figure 3. Rpb2 Wedge residues: structure, conservation, and function

A. Interaction of wedge residues with the minor groove of the upstream duplex. Sequence conservation of tip residues across species is nearly universal for Gly⁸⁶⁷ whose amide-bond interacts with the phosphate chain of the non-template strand; Met⁸⁶⁸ is conserved in yeast (S. cerevisiae and S. pombe) and is substituted by a bulky hydrophobic residue in other species. A refined 2F_obs-F_calc map contoured at 1.0σ is also illustrated. B. Conservation of wedge structure: yellow, T. thermophilus (Vassylyev et al., 2007); hot-pink, archaea (S. sulfactarius) (Hirata et al., 2008); sand, S. cerevisiae; blue, S. pombe (Spahr et al., 2009); and purple, 14-subunit Pol I (S. cerevisiae) (Engel et al., 2013). C. Overlay of mt-RNAP-upstream duplex (PDB:ID 4BOC) with our structure about template strand i+1 and i+2 (see also Fig. S3). D. In vitro elongation rate of wt Pol II and an rpb2 wedge deletion mutant (K864G/K865G/Δ866–871). Elongation rate determined on nucleic acid scaffolds at a number of NTP concentrations followed by non-linear regression of the rates for determination of maximum elongation rates (bar graph, error bars indicate range of 95% confidence interval). E. In vivo apparent elongation rates for wt Pol II and the rpb2 wedge deletion mutant at a galactose inducible reporter gene determine by chromatin IP upon glucose shutoff of transcription (schematic of reporter in F). Values are normalized to 0 minutes of glucose and error bars represent standard deviation of the mean for three independent experiments. F. Steady state occupancy for wt Pol II and the rpb2 wedge deletion mutant at a galactose inducible reporter gene under galactose induction determined by chromatin IP (schematic of reporter with positions of PCR amplicons shown below) (n=3 independent experiments). G. Steady state RNA levels of reporter used in F for wt Pol II and the rpb2 wedge deletion mutant. Values were normalized to SCR1 levels (a Pol III transcript) and averaged (n=3) with error bars representing standard deviation. H. Primer extension analysis for ADH1 transcripts in rpb2 wedge alleles (left) with quantification on right showing average change in fraction of ADH1 starts in various positions relative to wild type, with error bars representing standard deviation of the mean (n=3).

Figure 4. Architecture of the upstream fork junction

A. Stereo-view of the tripartite coordination of the three nucleic acid chains. Arch residues at the back, top and bottom adopt unique conformations –with respect to apo- and elongation structures– that interact with template, non-template and RNA strands respectively. Rpb1 residue Arg³²⁰ forms a H-bond with the 2′-OH of the nascent transcript at position i-8. B,C. Front view (B) and side view (C) of the upstream (closing) end of the bubble. Rudder (silver) and FL1 (sand) residues reach within 4 Å across the midline to form an arch that provides a scaffold for template and non-template strand annealing at i-12. Contacts include packing interactions between template strand i-8 and Tyr⁴⁵⁹ and potential H-bonds between Rpb2 residues Thr⁴⁶³ with template strand i-8, Glu⁴⁶⁹ with template strand i-10 and Lys⁴⁷¹ with non-template strand i-9. Lys³¹⁷ participates in contacts with non-template strand i-11 and i-12 (see also Fig. S4)

Figure 5. Architecture of the downstream fork junction

A. Stereo-view of the architecture of the downstream fork. Relevant interactions include U-loop residues with non-template strand i+5 and i+6, FL2 residues with non-template strand i+2 to i+3, and Rpb2 residues 221–282 (forming a 5 strand β-sheet, Dome) with non-template strand i+1 to i-2. B. Unbiased F_obs-F_calc electron density map contoured at 3σ (FL2 residues 498–512 were not included in map calculation). C. Structural overlay of published FL2 conformations during different stages of transcription. Δ4/7-TC (sand), PDB:ID 1Y1W (blue, elongation complex), PDB:ID 3HOW (cyan, backtracked complex), and PDB:ID 3PO2 (red, backtracked complex). D. Representative spectra of 2-AP probes at i+2 or i+3, bound to complementary non-template strand in the absence (open red circles and squares, respectively) and presence (blue circles and squares, respectively) of Pol II. The excitation wavelength was 315 nm and the fluorescence emission (shown in counts per second ×10⁶) was collected from 340–400 nm. E. Normalized fluorescence values for polymerase bound to ssDNA (primer-template) where 2AP is at the i+2, i+3, i+5 or i+8 position (red) and polymerase bound to dsDNA (primer-template annealed to fully complementary non-template strand) (blue). Error bars are standard deviation of the mean (n=3, see also Fig. S5).

Figure 6. Trigger loop (TL) and nucleic acid scaffold interactions during translocation

On-, off- state residues will be indicated in blue and grey, respectively. A modeled UTP (PDB:ID 2NVZ) is indicated in light grey/orange. A. Conformational changes observed between off and on (structural overlay with PDB:ID 2NVZ) states of the TL and Rpb1 funnel helices. A 2F_obs-F_calc map rendered at 1.0 σ is contoured around TL and funnel residues. B. Conformational changes observed between TL off and on states. Red arrows indicate motion. During off/on state conformational changes, most TL stabilizing interactions are disrupted, including: 1) Release of Met¹⁰⁷⁹ from its hydrophobic pocket. 2) Disruption of α2-Bridge Helix H-bonds, resulting in bridge helix displacement. 3) Disruption of Thr¹⁰⁹⁵-Thr¹¹¹³ H-bonds allowing counterclockwise TL motion. 4) Disruption of non-template strand - U-loop bonds, possibly leading to non-template strand release and translocation. C. Mutations of residues that disrupt Met¹⁰⁷⁹ hydrophobic pocket result in gain of function phenotypes (Kaplan et al., 2012). TLB residues are represented as a solid silver surface. Motion of Met¹⁰⁷⁹ might occur through a defined pathway on the protein surface (orange trace). Mutations of Ala¹⁰⁷⁶ and Gly¹⁰⁹⁷ (red surface) for bulkier residues, can potentially disrupt the vestibule of the hydrophobic pocket (Kireeva et al., 2012). D. Possible coupling of the global translocation of the scaffold to local motion of the TL. Pol II regions in contact with upstream and downstream duplexes, Rpb2 (sand) and Rpb5 (magenta), respectively, are coupled through TLB residues (dark gray). TL off and on conformations are illustrated in yellow and blue, respectively (see also Fig. S6).