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. 2014 May 27;111(21):7665-70.
doi: 10.1073/pnas.1315751111. Epub 2014 Apr 21.

Millisecond dynamics of RNA polymerase II translocation at atomic resolution

Daniel-Adriano Silva  1 , Dahlia R Weiss  2 , Fátima Pardo Avila  1 , Lin-Tai Da  1 , Michael Levitt  3 , Dong Wang  4 , Xuhui Huang  5
Affiliations

Millisecond dynamics of RNA polymerase II translocation at atomic resolution

Daniel-Adriano Silva et al. Proc Natl Acad Sci U S A. .

Abstract

Transcription is a central step in gene expression, in which the DNA template is processively read by RNA polymerase II (Pol II), synthesizing a complementary messenger RNA transcript. At each cycle, Pol II moves exactly one register along the DNA, a process known as translocation. Although X-ray crystal structures have greatly enhanced our understanding of the transcription process, the underlying molecular mechanisms of translocation remain unclear. Here we use sophisticated simulation techniques to observe Pol II translocation on a millisecond timescale and at atomistic resolution. We observe multiple cycles of forward and backward translocation and identify two previously unidentified intermediate states. We show that the bridge helix (BH) plays a key role accelerating the translocation of both the RNA:DNA hybrid and transition nucleotide by directly interacting with them. The conserved BH residues, Thr831 and Tyr836, mediate these interactions. To date, this study delivers the most detailed picture of the mechanism of Pol II translocation at atomic level.

Keywords: Markov state model; molecular dynamics; trigger loop.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
(A) RMSD of the active site with respect to the posttranslocation state as a function of time for the 1-ms MSM simulation trajectory (Materials and Methods). The segment between 10 and 35 μs is shown in the insert. The average RMSD for states 1–4 is shown by four dashed lines colored black, cyan, orange, and green for states 1–4, respectively. (B–E) Representative conformations of the metastable states identified by our model. Detailed views of the active site are shown for each of the four states identified by our model. Nucleic acids, ions, Thr831, and Tyr836 are shown in a sphere representation, whereas protein elements are shown in cartoon representation. The RNA (red), template DNA strand (cyan), BH (green), TL (purple), Mg2+ A (magenta), Rpb1 Thr831 (wheat), Rpb1 Tyr836 (yellow), and TN (orange) are shown. (B) State 1 (S1) corresponds to the pretranslocation state, analogous to the one found in X-ray crystallographic studies. (C and D) States 2 and 3 are intermediates of the translocation identified by the MSM. (E) State 4 also corresponds to the posttranslocation state found in X-ray crystallographic studies.
Fig. 2.
Fig. 2.
Translocation of the RNA:DNA hybrid is not synchronous with the translocation of TN. (A) A cartoon of the four metastable states, S1 to S4, shows the order of backbone and nucleotide translocation. From S1 to S2 the backbones of the RNA:DNA hybrid (red and cyan) translocate from pretranslocation to posttranslocation positions, whereas the TN (orange) lags behind and stacks with the BH (green). From S2 to S3 the TN crosses over the BH toward the active site but still remains stacked with the BH. From S3 to S4 the TN moves into the active site, losing the stacking with the BH and completing the translocation. (B) The plots show the distance to position in posttranslocation state for the backbone phosphate versus distance to position in the posttranslocation state for the nucleotide base. For DNA (Top) and RNA (Middle) the values are averaged over the eight upstream nucleotides of the DNA and RNA (also see Fig. S3), respectively. For the DNA TN (Bottom) there is just one phosphate and one base, so no averaging is needed.
Fig. 3.
Fig. 3.
The dynamics of the BH and TL direct the translocation. (A) The BH motion projected along the alpha helix axis shows that the bending can be as large as 10 Å, with a preference to bend toward the upstream RNA:DNA hybrid (see Fig. S5 for details of the projection). The cyan and orange dashed ovals illustrate the i+1 and i+2 template DNA positions. (B) The RMS fluctuation values of the Cα atoms in the BH show that its maximum bending occurs in the middle of the helix. (C) In the pretranslocation state (S1) the helix can bend enough to interact with the base in the i+1 position, and the correlation plots show that indeed the movement of the helix is correlated to the displacement of the i+1 DNA position (blue). However, in both intermediates the TN motion is tightly correlated with the BH. The first intermediate (S2) centered on the residues next to the Tyr836 and the second intermediate (S3) centered with the residues surrounding the Thr831. Finally, in the posttranslocation (S4), most of the correlation is lost. (D) The graph of the cross-correlation between the TL and the BH Cα atoms (Left) shows that the motion of the TL in these segments is highly correlated to the motion of the BH.
Fig. 4.
Fig. 4.
Stacking between BH residue Tyr836 and the TN base plays an important role in facilitating translocation. (A) A representative structure from state 2 shows π-stacking between Tyr836 (yellow) and the TN base (orange). Such stacking is not seen in the X-ray structures of Pol II either before (PDB ID: 1I6H) or after translocation (PDB ID: 2E2H). (B) Multiple sequence alignment across different species shows conservation of Tyr836, Thr831, and Gly835. These same residues show highly correlated motion to the BH and the TN. (C) Mutant simulations reveal that disrupting the π-stacking interaction between Tyr836 and the TN hinders forward translocation to the posttranslocation state.
Fig. 5.
Fig. 5.
The schematic free-energy landscape of translocation. The pathway from the pretranslocation state S1 (red curve) to the posttranslocation state S4 (green curve) has two metastable intermediates (S2 and S3; blue and orange curves). Representative structures of the states are displayed together with their equilibrium populations and the average times (in μs) for transitioning between them. The transition from S1 to S2 is rate-limiting. All of the major pathways connecting S1 and S4 have to go through S2 (see Supporting Information for details of the pathway analysis).
Fig. 6.
Fig. 6.
A model of Pol II translocation. At the pretranslocation state (S1), the oscillation of the BH is large enough to interact with the i+1 DNA nucleotide, which can then facilitate the motion of the RNA:DNA hybrid toward the posttranslocation state. At the first intermediate state (S2), the backbone of the upstream RNA:DNA hybrid has been translocated, whereas the TN still lags behind, stabilized by a stacking interaction with Tyr836. The active site is empty, which may permit entrance of the incoming NTP to the i site. At the second intermediate state (S3), the continuous oscillation of the BH further facilitates TN crossing over it, while maintaining strong interactions, mainly through residue Thr831. The position of the TN in S3 may already allow partial interaction with the incoming NTP. In the final steps, the TN moves to its final i+1 posttranslocation position (S4). The incoming NTP may then lock the system in the posttranslocation state by entering to the i site. The transparent surface surrounding the BH represents its overall displacement in each state. The BH residues that have correlated motion with the i+1 DNA nucleotide, TN, and both of them (i+1 DNA nucleotide and TN) are displayed in blue, green, and turquoise, respectively. The thickness of arrows that connect different states is proportional to the rate of the transitions between them.
See this image and copyright information in PMC

Comment in

  • Unveiling translocation intermediates of RNA polymerase.
    Imashimizu M, Kashlev M. Imashimizu M, et al. Proc Natl Acad Sci U S A. 2014 May 27;111(21):7507-8. doi: 10.1073/pnas.1406413111. Epub 2014 May 14. Proc Natl Acad Sci U S A. 2014. PMID: 24828529 Free PMC article. No abstract available.

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