Kinetics of nucleotide entry into RNA polymerase active site provides mechanism for efficiency and fidelity

Abstract

During transcription, RNA polymerase II elongates RNA by adding nucleotide triphosphates (NTPs) complementary to a DNA template. Structural studies have suggested that NTPs enter and exit the active site via the narrow secondary pore but details have remained unclear. A kinetic model is presented that integrates molecular dynamics simulations with experimental data. Previous simulations of trigger loop dynamics and the dynamics of matched and mismatched NTPs in and near the active site were combined with new simulations describing NTP exit from the active site via the secondary pore. Markov state analysis was applied to identify major states and estimate kinetic rates for transitions between those states. The kinetic model predicts elongation and misincorporation rates in close agreement with experiment and provides mechanistic hypotheses for how NTP entry and exit via the secondary pore is feasible and a key feature for achieving high elongation and low misincorporation rates during RNA elongation.

Keywords: Markov state model; Molecular dynamics simulation; NTP discrimination; Network model; Secondary pore.

Figure 1. RNA polymerase II structure, electrostatics, and secondary pore

10-subunit S. cerevisiae Pol II complex with open trigger loop and the three initial UTP sites (shown as spheres colored yellow, blue, and pink) that were used as the starting models in this study. Colors distinguish different subunits (Rpb1–4, 5–6, 8–12) and nucleic acid components (A). Electrostatic potential in the secondary pore projected onto the molecular surface according to the Poisson-Boltzmann equation calculated via APBS [30] with red and blue colors indicating negative and positive potentials respectively (B). Secondary pore with the nascent RNA shown in yellow, residues lining the pore used to define the NTP position within the pore (cf. Section 2.1) in blue, and the trigger loop in red (C).

Figure 2. NTP exit pathways via secondary pore from molecular dynamics simulations

Secondary pore exit pathways based on NTP center of mass positions (shown as black, red, and blue spheres) from 100 RAMD simulations projected onto the initial structure of Pol II. The trigger loop is shown in cartoon representation and colored corresponding to each pathway. Percentages indicate the relative occurrence of each pathway (A). Macrostates from MSM shown as UTP orientations within the secondary pore of the initial structure of Pol II. Conformations grouped into superstates are colored as follows: E, blue; G, green; T, purple; F, brown; H, tan; and bulk, grey (B). PMF for NTP positions in the secondary pore from snapshots of unbiased simulations as a function of the distance from the active site and projection onto the Cα D481→Cα T831 vector. Binding states used in the kinetic model (Fig. 3) are indicated as letters (C).

Figure 3. Kinetic network model and predicted elongation and misincorporation rates

Kinetic network model capturing NTP entry and RNA elongation with the main model represented by solid gray lines and alternate, hypothetical NTP entry pathways indicated as dashed lines (A). Elongation (black solid line and colored dashed lines; left scale) and misincorporation (dotted lines; right scale) rates as a function of bulk NTP concentration predicted from kinetic model. Black lines show results for full kinetic network model. Red, blue, and green lines correspond to alternative models where NTPs directly enter A, E, or G sites, respectively. Experimental pause-free elongation rates from single-molecule study of Pol II [40] are shown as black stars (B).