Multi-subunit RNA polymerases bind nucleotide triphosphate (NTP) substrates in the pretranslocated state and carry the dNMP-NTP base pair into the active site for phosphoryl transfer. NTP-driven translocation requires that NTP substrates enter the main-enzyme channel before loading into the active site. Based on this model, a new view of fidelity and efficiency of RNA synthesis is proposed. The model predicts that, during processive elongation, NTP-driven translocation is coupled to a protein conformational change that allows pyrophosphate release: coupling the end of one bond-addition cycle to substrate loading and translocation for the next. We present a detailed model of the RNA polymerase II elongation complex based on 2 low-affinity NTP binding sites located in the main-enzyme channel. This model posits that NTP substrates, elongation factors, and the conserved Rpb2 subunit fork loop 2 cooperate to regulate opening of the downstream transcription bubble.

Strategies for assembly and analysis of human, yeast and bacterial RNA polymerase elongation complexes are described, and methods are shown for millisecond phase kinetic analyses of elongation using rapid chemical quench flow. Human, yeast, and bacterial RNA polymerases function very similarly in NTP-Mg 2+ commitment and phosphodiester bond formation. A "running start, two-bond, double quench" protocol is described and its advantages discussed. These studies provide information about stable NTP-Mg 2+ loading, phosphodiester bond synthesis, the processive transition between bonds, and sequence-specific effects on transcription elongation dynamics.

When nucleoside triphosphate (NTP) substrates and ␣-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 ␣-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.A goal of transient state kinetic analyses is to reveal the internal mechanism of an enzyme reaction by observing synchronized millisecond events on a millisecond time scale. In this work, the mushroom toxin ␣-amanitin is utilized as a transient state inhibitor, locking human RNA polymerase II in the core of the elongation mechanism. By altering the conditions of stalling and by employing two distinct reaction quenching methods, details of the core mechanism of human RNA polymerase II become apparent. 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 (designated iϩ1). Very little space is available in these structures to load NTPs through the main enzyme channel, which holds the DNA duplex and RNA-DNA hybrid. The downstream transcription bubble is closed at the iϩ2 position by base pairing, so it is difficult to imagine how NTP substrates could interact at iϩ2 or iϩ3 template sites. T. thermophilus ␤ Arg 422 makes a specific contact to the iϩ1 DNA template phosphate, and appears to provide a specific mechanism for closing the downstream bubble at the iϩ2 position. This mechanism for closing the downstream bubble is not conserved in human or yeast RNA polymerase II, in which the corresponding residue to ␤ Arg 422 is Rpb2 Gly 493 (human) and Rpb2 Gly 506 (yeast). Furthermore, even if the downstream bubble were open in the T. thermophilus structure, little space is available to...

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