Fig 1.
Structural model of Pol II elongation complex with the complete transcription bubble.
The left panel shows a cut-view of our Pol II model with a complete transcription bubble. The protein is shown in grey color (surface representation), the bridge helix is shown in dark green color (cartoon representation) and the magnesium ion is shown by a purple sphere. The DNA template, DNA non-template and mRNA strands are shown in cyan, green and red colors, respectively. The right-top panel shows the whole model without the cut view, with the various subunits of Pol II shown in different colors. The right-bottom panel is a close-up of the transcription bubble shown in dark blue.
Fig 2.
Structural validation of the model by MD simulations: Nucleic acids flexibility.
(a) The scheme represents the nucleic acid scaffold used in our simulation (numbers denote nucleotide positions). (b)-(d) The RMSF, per nucleotide position, observed in our MD simulations for: mRNA, template DNA and non-template DNA. Grey background denotes those nucleotides that have been observed in crystallographic structures.
Fig 3.
Structural validation of the model by MD simulations: Base-pairing stability.
The bar plots show the probability of finding the optimal number of hydrogen bonds for: (a) between the template and non-template DNA strands, and (b) between the DNA-RNA hybrid. In the middle, the nucleic acid scaffold scheme is demonstrated with the region where the DNA duplex opens to form the transcription bubble shown in dark blue.
Fig 4.
The secondary channel exhibits higher probability for NTP entry than the main channel.
The cavity analysis was performed using the program CAVER (see Methods) with an ensemble of MD conformations of the elongation complex (without NTP). (a) The most possible pathways with enough space to allow the pre-loading of a NTP to the i+2 site are shown in yellow. Similarly, (b) shows the most possible pathways for a NTP to reach the active site in orange. In (c), the bar plot shows the probability of finding MD conformations with enough space for NTP diffusion through the main channel versus the secondary channel (see Methods). The template DNA (cyan), non-template DNA (green) and RNA (red) strands are shown with tube and licorice representations. The protein components are shown in grey with a cut view with a surface representation.
Fig 5.
MD relaxation of NTP in the main channel and the secondary channel.
(a-b) Initially, the points constituting the entry routes through the main and secondary channels were divided into 20 clusters. The yellow and orange spheres represent the center of each cluster for the main channel and the secondary channel, respectively. (c-d) A NTP molecule (in licorice presentation) accompanied by a bound magnesium atom (sphere representation) was aligned to each of the cluster centers (shown in (a-b)) by its center of mass (see Methods). These conformations were used as starting points for relaxation via MD simulations (see Methods). The template DNA, non-template DNA and mRNA strands are shown in cyan, green and red, respectively, with tube and licorice representations. The cut-view of the protein is shown in grey with a surface representation.
Fig 6.
Non-bonded interactions NTP experiences revealed by MD.
The plots compare the energetic contribution of non-bonded interactions (electrostatic + vdW) along the main channel and secondary channel for: (a) NTP and its environment (the Pol II complex and solvent); (b) NTP and the Pol II complex; and (c) NTP and solvent. In these calculations, the Pol II complex includes the protein and nucleotides. The solvent contains water molecules together with the counter-ions. The results shown in (a) are the total contributions from the Pol II complex (shown in (b)) and the solvent (shown in (c)) (see Methods).
Fig 7.
Distribution of charges surrounding the NTP during MD simulations.
(a) The plot shows the net charge of the Pol II (protein and nucleotides, y-axis) within certain radius (x-axis) of the NTP in the diffusion pathways through the main channel (dark grey) or the secondary channel (light grey). (b)-(d) The same as in (a) but for the charges of the nucleotides, positively-charged amino acids (a.a.) and negatively-charged a.a. around NTP, respectively.