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. 2021 Nov 15:8:772486.
doi: 10.3389/fmolb.2021.772486. eCollection 2021.

Modeling DNA Opening in the Eukaryotic Transcription Initiation Complexes via Coarse-Grained Models

Genki Shino  1 , Shoji Takada  1
Affiliations

Modeling DNA Opening in the Eukaryotic Transcription Initiation Complexes via Coarse-Grained Models

Genki Shino et al. Front Mol Biosci. .

Erratum in

Abstract

Recently, the molecular mechanisms of transcription initiation have been intensively studied. Especially, the cryo-electron microscopy revealed atomic structure details in key states in the eukaryotic transcription initiation. Yet, the dynamic processes of the promoter DNA opening in the pre-initiation complex remain obscured. In this study, based on the three cryo-electron microscopic yeast structures for the closed, open, and initially transcribing complexes, we performed multiscale molecular dynamics (MD) simulations to model structures and dynamic processes of DNA opening. Combining coarse-grained and all-atom MD simulations, we first obtained the atomic model for the DNA bubble in the open complexes. Then, in the MD simulation from the open to the initially transcribing complexes, we found a previously unidentified intermediate state which is formed by the bottleneck in the fork loop 1 of Pol II: The loop opening triggered the escape from the intermediate, serving as a gatekeeper of the promoter DNA opening. In the initially transcribing complex, the non-template DNA strand passes a groove made of the protrusion, the lobe, and the fork of Rpb2 subunit of Pol II, in which several positively charged and highly conserved residues exhibit key interactions to the non-template DNA strand. The back-mapped all-atom models provided further insights on atomistic interactions such as hydrogen bonding and can be used for future simulations.

Keywords: DNA opening; eukaryotes; molecular dynamics simulation; protein-DNA complex; transcription.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Three yeast PICs of RNA polymerase II and the promoter sequence used. (A) The three PICs obtained by cryo-EM; the closed complex (CC) (PDB: 5FZ5) (left), the open complex (OC) (PDB: 5FYW) (center), and the initially transcribing complex (ITC) (PDB: 4V1N) (right). The CC and OC models contain the promoter DNA, Pol II, TBP, TFIIA, TFIIB, TFIIE, and TFIIF. The ITC model contains the promoter DNA, Pol II, TBP, TFIIB, and 6 bp nascent RNA. Parts of the melted DNA were not modeled in the OC and ITC states (Red and orange broken lines). (B) The promoter DNA sequence used in the current study (numbered relative to the transcription starting site). The sequences are taken from those used in the cryo-EM studies of the OC and the ITC (Plaschka et al., 2016; Plaschka et al., 2015). Blue, the template DNA strand; cyan, the non-template DNA strand; red dashed square, TATA box; gray square, region to which mismatched sequence is introduced in a simulation; green and blue horizontal dashed lines along the sequence, the regions not appeared in the OC and ITC models by cryo-EM, respectively.
FIGURE 2
FIGURE 2
Coarse-grained MD simulation for the transition from the CC to OC states. Results of a representative trajectory are shown. (A) Snapshots at 0 MD step (left, the CC state), at 50 × 104 MD steps (center, the pre-OC state), and at 500 × 104 MD steps (right, the OC state with the mismatch). Some proteins are not displayed to make DNA visible. The same colors are used as Figure 1A. Blue and cyan region in DNA indicates the 15-bp mismatch region, forming the DNA bubble. (B) The time course of the fractions of protein-DNA contacts specific to CC (red) and OC (green). (C) The time course of the DNA bubble size. In (B,C), the left/right panels are from the first/second halves of MD simulations with/without the DNA mismatch. The blue curve in the right panel in (C) shows a moving average over 11 points.
FIGURE 3
FIGURE 3
The open DNA in the OC state of PIC. (A) Atomic structure model for the OC state. Some proteins are not displayed to make the DNA visible. (B) A close-up view of the orange dashed squared area in (A). Pink, the E-wing of TFIIE; yellow dashed lines, hydrogen bonds between DNA and the E-wing. (C) Open DNA structures with the bubble size of 6 bp (left) and 9 bp (right). Orange, the template DNA strand in the bubble.
FIGURE 4
FIGURE 4
Coarse-grained MD simulation for the transition from the OC to ITC states. Results of two representative trajectories are shown in red and blue curves. (A) Snapshots from the red trajectory at 0 MD step (top, the OC state), at 500 × 104 MD steps (I1 state), at 1500 × 104 MD steps (the I2 state), and 2000 × 104 MD steps (bottom, the ITC state). (B) The time course of the fractions of protein-DNA contacts specific to ITC. (C) The time course of the DNA bubble size. (D) The time courses of the distance between the centers of mass of the fork loop 1 of the Pol II Rpb2 (468–476 residues) and the B-linker in TFIIB (99–102 residues). Green dashed lines, a characteristic distance for the template DNA to pass through the fork loop 1.
FIGURE 5
FIGURE 5
The open DNA in the intermediate state I2. (A) The atomic structure model for the I2 state. Some proteins are not displayed to make the DNA visible. (B) The close-up view of the fork loop 1 (pale green) that blocks the template DNA passage. (C) Multiple sequence alignment of the fork loop 1 region of the Rpb2. Green, invariant; yellow, conserved.
FIGURE 6
FIGURE 6
The open DNA in the ITC state of PIC. (A) The atomic structure model for the ITC state. Some proteins are not displayed to make DNA visible. (B) The atomic structure model from the back side of (A), which focuses the non-template DNA strand path. (C) The close-up view of the Rpb2 and non-template DNA strand in the squared area in (B). Blue, positively charged residues; pink, I251 and S474 that form hydrogen bonds to DNA; yellow dashed lines, hydrogen bonds between bases of the non-template DNA strand and amino acids. (D) Multiple sequence alignment of the residues around those shown in (C). Green, invariant; yellow, conserved.
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