Role of bacterial RNA polymerase gate opening dynamics in DNA loading and antibiotics inhibition elucidated by quasi-Markov State Model

Abstract

To initiate transcription, the holoenzyme (RNA polymerase [RNAP] in complex with σ factor) loads the promoter DNA via the flexible loading gate created by the clamp and β-lobe, yet their roles in DNA loading have not been characterized. We used a quasi-Markov State Model (qMSM) built from extensive molecular dynamics simulations to elucidate the dynamics of Thermus aquaticus holoenzyme's gate opening. We showed that during gate opening, β-lobe oscillates four orders of magnitude faster than the clamp, whose opening depends on the Switch 2's structure. Myxopyronin, an antibiotic that binds to Switch 2, was shown to undergo a conformational selection mechanism to inhibit clamp opening. Importantly, we reveal a critical but undiscovered role of β-lobe, whose opening is sufficient for DNA loading even when the clamp is partially closed. These findings open the opportunity for the development of antibiotics targeting β-lobe of RNAP. Finally, we have shown that our qMSMs, which encode non-Markovian dynamics based on the generalized master equation formalism, hold great potential to be widely applied to study biomolecular dynamics.

Keywords: Markov State Model; bacterial RNA polymerase; transcription initiation.

Fig. 1.

qMSM identifies four metastable states corresponding to the opening and closing of RNAP clamp domain. (A) The overall cartoon representation of bacterial holoenzyme and its domains, clamp, β-lobe, and Switch 2 region are shown as yellow, magenta, and orange, respectively. (B) Four macrostates of the clamp domain of RNAP are identified by MSM. The metastable states are represented as clamp opening/closing angles and rmsd of Switch 2 region (β’611 to 620) aligned to the Switch 2 region of the closed clamp in the crystal structure. The clamp opening/closing angle ($\text{Θ}$) is defined as the torsion angle between four centers of mass of C-α of residues 1) ω 60 to 68, 2) β’1461 to 1468, 3) β1031 to 1033 and β’620 to 622, and 4) β’568 to 573 and is offset by the angle of closed clamp crystal structure ($\text{Θ}-{\text{Θ}}_{\text{closed}}$). (C) The transition time between metastable states and the stationary population of each state is shown. The transitions between S1 and S2, as well as S3 and S4, are fast. The transitions between S1 or S2 to either S3 or S4 are slow, as indicated by the higher free-energy barrier. The full list of transition times is shown in SI Appendix, Table S1.

Fig. 2.

Switch 2 region under the clamp acts as a hinge of the clamp domain opening and closing. (A) Switch regions are located under the clamp domain. There are three switch regions, Switch 1, Switch 2, and Switch 3, which are adjacent to each other. The viewing angle of the switch regions is shown by eye symbol in the left figure. (B) Diameter of the α-helix of the Switch 2 region in each metastable state is shown. The viewing direction of these figures is perpendicular to the viewing direction of A and into the protein core. The diameter is the distance between center of mass of C-α atoms of residue β’615 and 619 and C-α atom of residue β’617. Small diameter of helix at ∼4.5 Å indicates α-helical conformation, while large diameter of helix at ∼5.2 Å indicates π-helical conformation. (C) Pearson’s correlation coefficient of the clamp opening/closing angle with the rmsd of Switch 1, 2, 3 regions aligned to the C-α atoms of the individual Switch regions in the closed-clamp crystal structure. For the calculation of rmsd of switch regions, the C-α atoms of the following residues are included: 1) β’1431 to 1443 for Switch 1, 2) β’610 to 620 for Switch 2, and 3) β1010 to 1032 for Switch 3. Based on correlation analysis, only conformational change of Switch 2 is strongly correlated with the clamp opening/closing motion. The error bars were obtained by bootstrapping of independent trajectories.

Fig. 3.

β-Lobe is a highly dynamic domain compared to clamp. (A) The open and closed conformations of β-lobe are shown as magenta and black, respectively. The β-lobe opening angle is defined as the dihedral angle formed by the center of mass of four groups of residues represented by the C-α atoms: 1) β’877 to 889, 2) β’938 to 943, 3) β’786 to 793, and 4) β142 to 325. (B) Distribution of β-lobe opening angle in each metastable state is shown. The open β-lobe state consists of structures having β-lobe opening angle larger than μ + 1.5σ, where μ and σ is the average and SD of all MD trajectories. The closed β-lobe state consists of the structures having β-lobe opening angle smaller than μ − 1.5σ. (C) Average transition time values of β-lobe opening in all the dataset and each metastable state. The opening of the β-lobe is two to three orders of magnitude faster than the clamp opening.

Fig. 4.

Spontaneous detachment or unfolding of Switch 2 region only occurs in a partially closed state, S2, which allows binding of Myx. (A) The structure of Switch 2 region when bound to Myx is shown. Switch 2, helix 1084 to 1090, helix β’1463 to 1467 are shown in orange, purple, and pink cartoon. Myx is shown as cyan sticks representation. D3 or D4 are the distance between center of mass represented by C-α atoms of β’617 to 620 (part of Switch 2) and β1084 to 1085 (D3) or β’1466 to 1467 (D4), respectively. (B) The free-energy landscape represented as D3 and D4 are shown for each metastable state. The plus sign (+) represents the cocrystal structure of RNAP bound with Myx (PDB ID: 3DXJ). The detached and folded Switch 2 structure in S2 is shown as (Δ) and ($\nabla$). (C) The docking pose of Myx to a folded ($\nabla$) Switch 2 is shown as lime sticks representations. (D) The docking pose of Myx to a detached (Δ) Switch 2 is shown as lime sticks representation, which also corresponds to the best docking pose (largest number of native contacts). In Fig. A , C, and D, and in both C and D, Myx in the cocrystal structure (PDB ID: 3DXJ) is overlaid and shown as cyan sticks representation. (E) Docking results of Myx to RNAP conformations in each metastable state are plotted as docking score and number of native contacts.

Fig. 5.

Opening of β-lobe is sufficient for DNA loading even when the clamp is partially closed. (A) D1 is the distance between center of mass of clamp and β-lobe. The atoms included for the calculation of center of mass is the same as the ones included for PCA. (B) Merging the clustering in A and B results in 12 combinations of clamp and β-lobe conformations. The D1 values for the structure closest to the cluster centers from each combination are shown as bar graph. There are three combinations of clamp and β-lobe conformation that can accommodate dsDNA, which are (C) open and open, (D) open and partially closed, (E) partially closed and open, respectively.