Complete architecture of the archaeal RNA polymerase open complex from single-molecule FRET and NPS

Abstract

The molecular architecture of RNAP II-like transcription initiation complexes remains opaque due to its conformational flexibility and size. Here we report the three-dimensional architecture of the complete open complex (OC) composed of the promoter DNA, TATA box-binding protein (TBP), transcription factor B (TFB), transcription factor E (TFE) and the 12-subunit RNA polymerase (RNAP) from Methanocaldococcus jannaschii. By combining single-molecule Förster resonance energy transfer and the Bayesian parameter estimation-based Nano-Positioning System analysis, we model the entire archaeal OC, which elucidates the path of the non-template DNA (ntDNA) strand and interaction sites of the transcription factors with the RNAP. Compared with models of the eukaryotic OC, the TATA DNA region with TBP and TFB is positioned closer to the surface of the RNAP, likely providing the mechanism by which DNA melting can occur in a minimal factor configuration, without the dedicated translocase/helicase encoding factor TFIIH.

Figure 1. Schematic representation of the global FRET network used to determine the complete architecture of the archaeal open complex with NPS

(A) All unknown antenna positions (green circles) and the five known satellite sites on the RNAP (dark red circles) are shown together with the corresponding attached dyes (A647 = Alexa 647; A555 = Alexa555; Dl550 = DyLight550; Dl650 = DyLight650). FRET efficien-cies were measured between pairs of satellite (acceptor) and antenna (donor) dyes (dotted black lines) and in between antennas (dotted red lines). In case of the latter measurements, one of the antenna positions had to be labelled with an acceptor, as indicated. (B) Cartoon depicting the mismatched (-3 to +1) viral SSV T6 promoter (tDNA in blue, ntDNA in cyan, TATA box in red), which is used throughout this study. Labelling sites on the DNA and on the transcription factors TBP, TFB and TFE are marked with a green star. Labelling sites on the RNAP are marked with a dark red star. Proteins contained within the archaeal OC are shown schematically. See also Supplementary Figures 1, 2 and 4.

Figure 2. Localisation of two positions on the tDNA strand in the archaeal OC

(A) Cartoon depicting the labelling positions on the tDNA strand which were localised using the NPS (colour coding according to Figure 1). (B) - (F) Framewise smFRET histograms used in the NPS localisation of tDNA(-9). Shown is the smFRET data from measurements between tDNA(-9) and Rpo7-V49 (B), Rpo7-S65 (C), Rpo2"-Q373 (D), Rpo1'-G257 (E) and Rpo5-K11 (F), respectively. The histograms were fitted with a double (B-C, F) or single (D-E) Gaussian distribution indicated by the black lines. In (B-C, F) the gray line represents the sum of the two Gaussian distributions. Results of the fits together with those obtained during localisation of tDNA(+3) are summarized in Table 1. (G) NPS results for the fluorescent probes attached to tDNA(+3) (pink) and tDNA(-9) (yellow). The X-ray structure of the archaeal polymerase of S. shibatae (PDB: 2WAQ) is represented as dark grey ribbon. Note, that at this confidence level, the credible volume of tDNA(-9) is divided into two areas, if the credible volume is drawn at higher confidence these two areas merge (see text for details). (H) Comparison of the NPS results to the eukaryotic open complex model. The X-ray structure of the yeast polymerase is represented as light grey ribbon, the tDNA is shown in blue and the ntDNA is shown in cyan. The corresponding eukaryotic bases for tDNA(+3) (green) and tDNA(-9) are encircled. The NPS credible volumes are in good agreement to the model. See also Supplementary Fig. 3.

Figure 3. Localisation of the ntDNA strand in the archaeal OC

(A) - (B) Side and Front view of the archaeal RNA polymerase including the NPS results for the fluorescent probes attached to ntDNA(+7) (brown), ntDNA(-1) (dark red), ntDNA(-5) (red), ntDNA(-7) (orange), ntDNA(-10) (yellow), ntDNA(-12) (green) and ntDNA(-14) (dark green, hardly visible). (C) Top view (slightly rotated for a clearer view) of archaeal RNA polymerase with selective volumes shown at a time for clarity. See also Supplementary Figures 2 and 6.

Figure 4. Localisation of the TATA DNA, TBP, TFB and TFE in the archaeal OC

(A) - (B) Side and front view of the archaeal RNA polymerase together with the NPS results for the fluorescent probes attached to ntDNA(-18) (dark cyan), ntDNA(-24) (dark blue), ntDNA(-30) (magenta), ntDNA(-37) (gold), TBP-S71 (purple), TFB-G262 (olive), TFE-G44 (yellow) and TFE-G133 (green). (C) Top view of archaeal RNA polymerase with only the NPS credible volumes TFE-G44 (yellow) and TFE-G133 (green) shown at a time to illustrate their proximity to the clamp coiled coil region of subunit Rpo1' (red) and to the stalk of the RNAP (black), respectively. (D) Alternative view obtained by a 90° rotation of the top view, showing the proximity of the NPS credible volume ntDNA(-18) (dark cyan) to the protrusion (brown) of subunit Rpo2". The NPS credible volume of ntDNA(-24) (dark blue) is situated further away from the RNAP surface compared to the position of ntDNA(-18). Also, the proximity of the NPS credible volumes TBP-S71 (purple) and TFB-G262 (olive) to the wall domain (beige) of subunit Rpo2" and to subunit Rpo12 (light blue) can be seen. The volume of TFB-G262 is made transparent for clarity. See also Supplementary Figures 6 and 7.

Figure 5. Model of the complete archaeal OC complex

Side, Front and Top view an alternate view obtained from the top view by a 90° rotation of the model of the open complex. DNA template and non-template strand are in blue and cyan, respectively. The transcription factors TBP, TFB and TFE are in purple, green and yellow, respectively. The ntDNA positions used for building the model are colour coded according to the NPS densities in Figures 3 and 4, namely ntDNA(-1), ntDNA(+7), ntDNA(-1), ntDNA(-5), ntDNA(-7), ntDNA(-10), ntDNA(-12), ntDNA(-14), ntDNA(-18), ntDNA(-24), ntDNA(-30) and ntDNA(-37). The X-ray structure of the archaeal polymerase of S. shibatae (PDB: 2WAQ37) is represented as dark grey ribbon. See also Supplementary Fig. 8.

Figure 6. Open complex model has implications for the melting of DNA in the CC to OC transition

(A) Side view (slightly rotated for a clearer view) of the archaeal polymerase of S. shibatae (PDB: 2WAQ) (dark grey) together with components of the open complex model, namely TFB, TBP as well as the template and non-template strand. (B) Detail of the yellow rectangular region in (A) showing the clamp coiled coil domain of subunit Rpb1' (light grey) and the point mutation introduced into the B-linker helix in a previous study (red). The rudder loop of Rpb1' is shown in pink. (C) - (D) Detailed views of the archaeal model compared to the eukaryotic open complex model. The eukaryotic open complex model is displayed as superposition to the archaeal model and the archaeal RNAP. The eukaryotic transcription factors TBP and TFIIB are shown in light pink and light green, respectively. The DNA is shown in light blue and the polymerase is represented as semi-transparent surface. For the OC model the same colour coding is used as in Figure 5.

Figure 7. Mechanisms of the closed (CC) to open complex (OC) transition in archaea and eukaryotes

During open complex formation the double-stranded promoter DNA is melted and the template DNA strand (tDNA) is loaded into the active site while the nontemplate strand (ntDNA) interacts with the RNAP clamp, and with TFE and TFIIE in archaea and eukaryotes, respectively (highlighted in orange). Concomitantly the entire complex – RNAP and initiation factors – undergoes large scale conformational changes. In archaea OC formation occurs spontaneously and is possibly driven by the torsional strain in the promoter DNA induced by the interaction network between initiation factors, RNAP and the promoter DNA elements. While the upstream BRE and TATA promoter elements are anchored to the PIC by TFIIB (green) and TBP (magenta), the downstream DNA interacts with the RNAP jaws. In the Pol II system OC formation is largely driven by the ATP hydrolysis-dependent activities of the TFIIH subunit ssl2 (red) which also induces a torsional strain by translocating the downstream promoter DNA in the upstream direction into the active site of RNAP.