Eukaryotic RNA polymerase II catalyzes the transcription of DNA into mRNA very efficiently and with an extremely low error rate with regard to matching base and sugar moiety. Despite its importance, little is known about how it discriminates against 2′-deoxy NTPs during the chemical reaction. To investigate the differences in the addition reactions of ATP and dATP, we used FF-MD and QM/MM calculations within a nudged elastic band approach, which allowed us to find the energetically accessible reaction coordinates. By converging the QM size, we found that 800 QM atoms are necessary to properly describe the active site. We show how the absence of a single hydrogen bond between the enzyme and the NTP 2′-OH group leads to an increase of the reaction barrier by 16 kcal/mol and therefore conclude that Arg446 is the key residue in the discrimination process.