Diffusion of nucleoside triphosphates and role of the entry site to the RNA polymerase II active center

Abstract

Nucleoside triphosphates (NTPs) diffuse to the active center of RNA polymerase II through a funnel-shaped opening that narrows to a negatively charged pore. Computer simulation shows that the funnel and pore reduce the rate of diffusion by a factor of approximately 2 x 10(-7). The resulting limitation on the rate of RNA synthesis under conditions of low NTP concentration may be overcome by NTP binding to an entry site adjacent to the active center. Binding to the entry site greatly enhances the lifetime of an NTP in the active center region, and it prevents "backtracking" and the consequent occlusion of the active site.

Fig. 1.

Path of NTP entry to the active center of a pol II transcribing complex. The structure of a transcribing complex (PDB ID code 1R9T) is shown in space-filling representation, sectioned along the length of the NTP entry path, and viewed en face (as in figure 2C of ref. 16). Backbone models of the DNA (template strand in blue, and nontemplate strand in green) and RNA (red) are shown. The nucleotide A and E sites are circled in yellow. An NTP bound to the E site is shown in orange.

Fig. 2.

Computer simulations of NTP diffusion. (A) Selected trajectories of NTP diffusion in the funnel and pore in the presence of electrostatic potential and in the absence (Upper) or presence (Lower) of the E site. Each trajectory begins near the entrance to the funnel (lower boundary of the plot, as indicated by the colored bar on the left). The entrance to the pore is indicated by the dashed line and color change of the bar. Locations of A and E sites are marked on the bar. (B) Normalized sum of many trajectories for the case of no electrostatic potential and no E site (blue line), electrostatic potential but no E site (dashed line), or both electrostatic potential and E site (solid black line). The probability (average across a 1-Å-thick section of the funnel or pore) of an NTP at a particular position (distance from the A site) is shown. The orientation is with the A site at the left and pol II surface at the right, as indicated on the colored bar beneath the plot. The average trajectory computed for a simple conical opening is also shown (green line).

Fig. 3.

Electrostatic potential in the funnel and pore. The value of the electrostatic potential in planes through the funnel and pore (bar on the left) in the orientations indicated (Inset) is displayed on a scale from –3 to +3 (red to blue, respectively, as shown in the bars beneath the images).

Fig. 4.

Equilibrium distribution of NTP in the funnel and pore calculated from the Boltzmann relation. The value of [NTP]_{funnel–pore}/[NTP]_solution is plotted (thick dashed line) as a function of position in the funnel and pore (indicated in the bar beneath the plot). The value of the electrostatic potential from which this ratio of NTP concentrations was derived (solid line) was computed by averaging over 1-Å-thick slabs of the solvent-accessible space region of the funnel and pore. For comparison, the normalized sum of trajectories for the case of no E site (dashed line in Fig. 2B) is reproduced here (thin dashed line).