Abstract
When nucleoside triphosphate (NTP) substrates and alpha-amanitin are added to a human RNA polymerase II elongation complex simultaneously, the reaction becomes stalled in the core of the bond synthesis mechanism. The mode of stalling is influenced by NTP substrates at the active site and at downstream sites and by transcription factor IIF (TFIIF) and TFIIS. NTP substrates templated at i+2, i+3, and i+4 downstream DNA sites can reverse the previously stable binding of an NTP loaded at the i+1 substrate site. Deoxy-(d)NTPs and NDPs (nucleoside diphosphates) do not substitute for NTPs at the i+2 and i+3 positions (considered together) or the i+4, i+5, and i+6 positions (considered together). The mode of stalling is altered by changing the number of downstream template sites that are accurately occupied by NTPs and by changing NTP concentration. In the presence of the translocation blocker alpha-amanitin, a steady state condition is established in which RNA polymerase II stably loads an NTP substrate at i+1 and forms a phosphodiester bond but cannot rapidly complete bond synthesis by releasing pyrophosphate. These observations support a role for incoming NTP substrates in stimulating translocation; results appear inconsistent with the secondary pore being the sole route of NTP entry for human RNA polymerase II, and results indicate mechanisms of dynamic error avoidance and error correction during rapid RNA synthesis.
Highlights
Recent x-ray crystal structures of Thermus thermophilus RNA polymerase elongation complexes indicate a simple thermal ratchet mechanism for elongation [1, 2], with nucleoside triphosphate (NTP)2 substrates loading through the secondary pore, a solvent accessible channel, to the active site
NTP-driven Translocation—In the presence of a translocation block (␣-amanitin), we demonstrate robust effects of NTPs that are accurately templated at adjacent downstream positions on the fate of a substrate NTP loaded and initially tightened in the iϩ1 active site of human RNA polymerase II
Neither improperly templated NTPs, nor accurately templated NDPs, nor accurately templated dNTPs show these effects, revealing chemical selectivity at multiple downstream positions. Because these results demonstrate simultaneous NTP occupancy of the active site and downstream sites, these results support the NTP-driven translocation model and appear inconsistent with the secondary pore being the sole route of NTP loading for human RNA polymerase II
Summary
Isomerization reversal experiments and quantification of data were done essentially as described [12]. To reactions in which TFIIS was added during the ATP running start, 20 M CTP and UTP were added during the preincubation to maintain the C40 stall position. In this protocol, CTP and UTP are diluted to a working concentration of 5 M by two equal volume mixings. When TFIIS was not present during the preincubation, CTP and UTP were omitted (except as specified in individual protocols). A linear response over a 100-fold exposure range is sufficient to detect all relevant transcripts. Structures were modified (iϩ and iϩ NTPs placed by modeling, DNA strands extended, non-template DNA placed), as described previously [10]
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