A tunable ratchet driving human RNA polymerase II translocation adjusted by accurately templated nucleoside triphosphates loaded at downstream sites and by elongation factors.
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.
Xiong
Yalin
Y
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319, USA.
Burton
Zachary F
ZF
D053959:Alpha-Amanitin / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D003854:Deoxyribonucleotides / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D012313:RNA / Q000096:biosynthesis*; D012319:RNA Polymerase II / Q000037:antagonists & inhibitors / Q000737:chemistry*; D012265:Ribonucleotides / Q000737:chemistry*; D019473:Transcription Factors, TFII / Q000737:chemistry*; D035602:Transcriptional Elongation Factors / Q000737:chemistry*
D053959:Alpha-Amanitin; D003854:Deoxyribonucleotides; D012265:Ribonucleotides; D019473:Transcription Factors, TFII; D035602:Transcriptional Elongation Factors; C018648:transcription factor S-II; D012313:RNA; D004247:DNA; D012319:RNA Polymerase II; C056604:transcription factor TFIIF