Dynamic error correction and regulation of downstream bubble opening by human RNA polymerase II

The nucleotide triphosphate (NTP)-driven translocation hypothesis posits that NTP substrates bind to templated DNA sites prior to translocation into the active site. By using millisecond phase kinetics, we demonstrate this prediction in three different ways. First, we show that, in the presence of the translocation blocker a-amanitin, NTPs (but not deoxynucleotide triphosphate [dNTPs]) templated at downstream sites (i + 2 and i + 3) dislodge an active site (i + 1) NTP, which was otherwise fated to complete bond synthesis. Second, we show that NTPs templated at i + 2 and/or i + 3 downstream sites suppress misincorporation errors. Third, we show that NTPs templated at downstream sites stabilize the posttranslocated elongation complex at a stall position. Therefore, at least two NTP substrates pair to DNA templated sites downstream of the active site. These results demonstrate the mechanisms of NTP loading and transcriptional efficiency and fidelity for human RNA polymerase II and indicate regulation of downstream bubble opening by NTPs.