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Transcription factors IIF and IIS and nucleoside triphosphate substrates as dynamic probes of the human RNA polymerase II mechanism


Transcription factors IIF and IIS and nucleoside triphosphate substrates as dynamic probes of the human RNA polymerase II mechanism



Journal of Molecular Biology 342(4): 1085-1099



ISSN/ISBN: 0022-2836

PMID: 15351637

DOI: 10.1016/j.jmb.2004.07.070

The mechanism for elongation catalyzed by human RNA polymerase II (RNAP II) has been analyzed using millisecond phase transient state kinetics. Here, we apply a running start, two-bond, double-quench protocol. Quenching the reaction with EDTA indicates NTP loading into the active site followed by rapid isomerization. HCl quenching defines the time of phosphodiester bond formation. Model-independent and global kinetic analyses were applied to simulate the RNAP II mechanism for forward elongation through the synthesis of two specific phosphodiester bonds, modeling rate data collected over a wide range of nucleoside triphosphate concentrations. We report adequate two-bond kinetic simulations for the reaction in the presence of TFIIF alone and in the presence of TFIIF+TFIIS, providing detailed insight into the RNAP II mechanism and into processive RNA synthesis. RNAP II extends an RNA chain through a substrate induced-fit mechanism, termed NTP-driven translocation. After rapid isomerization, chemistry is delayed. At a stall point induced by withholding the next templated NTP, RNAP II fractionates into at least two active and one paused conformation, revealed as different forward rates of elongation. In the presence of TFIIF alone or in the presence of TFIIF+TFIIS, rapid rates are very similar; although, with TFIIF alone the complex is more highly poised for forward synthesis. Based on steady-state analysis, TFIIF was thought to suppress transcriptional pausing, but this view is misleading. TFIIF supports elongation and suppresses pausing by stabilizing the post-translocated elongation complex. When THIS is present, RNA cleavage and transcriptional restart pathways are supported, but THIS has a role in suppression of transient pausing, which is the most important contribution of THIS to elongation from a stall position.

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Accession: 018189015

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