(A) Pol I can bind the incoming nucleotide (GMPCPP) in the active site, while nucleotides of the opposite, non-template strand (+1 and+2), interact with the Fork loop two and Loop B. (B) GMPCPP is bound by conserved, identical residues in Pol I and Pol II. These include two arginines that interact with the phosphate (R714 and R957), a leucine from the trigger loop that stacks against the DNA base (L1202), and R591 and N625 which recognize the 2’- and 3’-OH groups, respectively. The ‘gating tyrosine’ (Y717), involved in RNA positioning during backtracking (Cheung and Cramer, 2011), and K916 and K924, which bind the 3’-end of the RNA are also indicated. Residues are shown in grey (A190) or tan (A135) for Pol I, while those in Pol II in dark green, and in Pol III in light green. Density for the DNA-RNA hybrid is from the sharpened, Pol I (core) EC (+GMPCPP) reconstruction, while the GMPCPP is from the same reconstruction but from the unsharpened/unmasked map. Density for L1202 is shown at a lower threshold. Residues are boxed according to their proposed role: black box, triphosphate binding; red box, nucleotide base stabilization; dashed box, NTP/dNTP discrimination. (C) Binding of GMPCPP is virtually identical in Pol I (top) and Pol II (bottom, PDB: 4a3j) (Cheung et al., 2011).( D) In the downstream edge of the transcription bubble, the +2 base of the NT strand is flipped into a pocket formed by Fork loop 2 (FL2) and loop B (‘A135 pocket’). These elements interact with the nucleotide through R219, R225 and the conserved D395. (E) These interactions also position the +1 base next to F508 from FL2 (top), resembling the interaction of the +1 base with βW183 in bacterial Pol (bottom, PDB: 6alh) (Kang et al., 2017). See also Figure 5—figure supplement 1.