The nucleotide selection scheme in T7 RNAP elongation. (Top) The free energy landscape for incorporating the right (solid line) and wrong (dashed) nucleotides in the five-state kinetic scheme. Four selection checkpoints (S₁toS₄) are labeled. Δ₁and Δ₂are differentiation free energies between the right and wrong nucleotides at the first two checkpoints. δ_Gis an overall free energy differentiation without polymerase. See Ref. [71] for details. (Bottom) Comparing the active site configurations when the right and wrong NTP bind respectively to the pre-insertion site.^[72]Y639 (red) is located on the C-term end of the Ohelix (cyan) to assist the nucleotide selection. Left: rATP (right) forms the Watson–Crick base pairing with the template. The recognition is assisted by water bridging HB interactions with Y639-OH and 2’-OH of rNTP. Right: dATP cannot base pair with the template due to the Y639 interference, which associates with dATP and stacks well with the DNA–RNA hybrid end, under water collision.^[72]