Mapping the route of nucleoside triphosphate (NTP) entry into the sequestered active site of RNA polymerase (RNAP) has major implications for elucidating the complete nucleotide addition cycle. Constituting a dichotomy that remains to be resolved, two alternatives, direct NTP delivery via the secondary channel (CH2) or selection to downstream sites in the main channel (CH1) prior to catalysis, have been proposed. In this study, accelerated molecular dynamics simulations of freely diffusing NTPs about RNAPII were applied to refine the CH2 model and uncover atomic details on the CH1 model that previously lacked a persuasive structural framework to illustrate its mechanism of action. Diffusion and binding of NTPs to downstream DNA, and the transfer of a preselected NTP to the active site, are simulated for the first time. All-atom simulations further support that CH1 loading is transcription factor IIF (TFIIF) dependent and impacts catalytic isomerization. Altogether, the alternative nucleotide loading systems may allow distinct transcriptional landscapes to be expressed. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/ ). Abstract Mapping the route of nucleoside triphosphate (NTP) entry into the sequestered active site of RNA polymerase (RNAP) has major implications for elucidating the complete nucleotide addition cycle. Constituting a dichotomy that remains to be resolved, two alternatives, direct NTP delivery via the secondary channel (CH2) or selection to downstream sites in the main channel (CH1) prior to catalysis, have been proposed. Keywords: RNA polymerase, nucleoside triphosphate, main channel, secondary channel, tertiary channel, downstream bubble, loading, diffusion, entry, TFIIF status released display-pdf yes is-olf no is-manuscript no is-preprint no is-journal-matter no is-scanned no is-retracted no Received 2020 Aug 7; Accepted 2020 Sep 3; Collection date 2020 Sep. 1. Introduction RNA polymerases (RNAPs) are nanoscopic machines responsible for transcribing sections of the information stored in DNA into RNA. In all cellular RNAPs, the catalytic site carrying out the RNA synthesis reactions is buried deep within a large multi-subunit protein complex. The evidence supporting direct delivery of individual NTPs to the active site via CH2 and the observations that NTPs can bind to several positions along the DNA template strand before transferring into the active site appear to be in contradiction. Mapping the NTP entry routes constitutes an arduous investigation because the diffusion process occurs in the sub-millisecond range and is an intrinsically dynamic process. Traditional experimental techniques, offering either limited spatial (e.g., transient state kinetics) or temporal (e.g., X-ray crystallography) resolutions, struggle to grasp the full mechanistic overview of the loading mechanism. The following amino/nucleic acids are shown in van der Waals spherical representation as follows: RNA i + 1 to i − 1 (medium blue); template DNA i + 2 to i − 1 (fade blue); bridge helix residues 1 E845 (fluo green), 1 D849 (green), 1 K853 (dark yellow); trigger loop residues 1 K1115, 1 P1122, 1 K1125, 1 E1126, 1 N1129 (orange); fork loop 2 residues 2 K494, 2 A496, 2 K497, 2 R499, 2 Q500 (grey); and key active site coordinating residues 2 R721 (pink), 2 R975 (red), 1 K775 (magenta). NTP bound magnesium ion, MgB, is shown as a silver sphere. Carboxy-terminal trigger base helix (TLc) 1 K1133/ 1 K1135 are displayed as transparent surfaces. The last on-pathway state consists of the transfer of the i + 2 isomerized NTP to the catalytic site. A transfer intermediate was detected in aMD_F6 and F’6, where the triphosphate portion shifted in the direction of bridge helix N-TER, and where MgB advanced near 1 E794/ 1 E845/ 1 D849. To further investigate the transfer process, the NTP isomerization configuration was aligned in a structure containing a bent bridge helix. Simulations aMD_G1, H2, I3, and J4 (aMD_H2, I3, and J4 were restarted from aMD_G1, H2, and I3, respectively) sampled a complete forward translocation step, accompanied with the transfer of the i + 2 NTP to the active site. The downstream template register shifted from the i + 2 to the i + 1 position, while the NTP molecule was shuttled to the other side of the bridge helix between the trigger loop and the F loop, and reassociated with the translocated DNA register ( Figure 7 E–I). During the process, up to 41 residues belonging to fork loop 2, trigger loop, bridge helix, F loop, F claw, link, and sleeve contact the NTP ( Figure S9 ). A very important conclusion that emerges from this simulation data is the confirmation that Significant elongation rate stimulation at very low NTP concentrations and kinetic switch to the rapid phase regime, in human and yeast RNAPII when supplemented with TFIIF (as well as in E. coli RNAP when comprising fork loop 2 required for i + 2 binding), have been reported in quench-flow kinetic and single-molecule optical trapping experiments [ 19 , 20 , 21 , 22 , 80 ]. Considering the correlation between the NTP loading mechanisms and the nucleotide addition cycle in more detail, the following hypothesis emerges: The CH1 mode of substrate loading appears to significantly support additional modes of the general transcription process by RNA polymerases. However, employing the CH2 mechanism, the right NTP must first successfully be selected after vacation of the active site before translocation oscillation would be adjourned. Fidelity. When the next NTP binds to i + 2, first, it was isomerized in CH3P ( Figure 7 B), then detached from template DNA ( Figure 7 F), and transferred to the catalytic site ( Figure 7 I) before undergoing final catalytic confinement. Therefore, there is a double isomerization process at play, utilizing the energy of base pairs twice, and delayed in time.