Chunfen Zhang

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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(More)
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(More)
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 alpha-amanitin, NTPs (but not(More)
Molecular dynamics simulation of Thermus thermophilus (Tt) RNA polymerase (RNAP) in a catalytic conformation demonstrates that the active site dNMP-NTP base pair must be substantially dehydrated to support full active site closing and optimum conditions for phosphodiester bond synthesis. In silico mutant beta R428A RNAP, which was designed based on(More)
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-Mg2+ commitment and phosphodiester bond formation. A(More)
When RNA polymerase II (RNAP II) is forced to stall, elongation complexes (ECs) are observed to leave the active pathway and enter a paused state. Initially, ECs equilibrate between active and paused conformations, but with stalls of a long duration, ECs backtrack and become sensitive to transcript cleavage, which is stimulated by the EC rescue factor(More)
The role of the RAP74 1 helix of transcription factor IIF (TFIIF) in stimulating elongation by human RNA polymerase II (RNAP II) was examined using millisecond-phase transient-state kinetics. RAP74 deletion mutants RAP74(1–227), which includes an intact 1 helix, and RAP74(1–158), in which the 1 helix is deleted, were compared. Analysis of TFIIF RAP74-RAP30(More)
The role of the RAP74 alpha1 helix of transcription factor IIF (TFIIF) in stimulating elongation by human RNA polymerase II (RNAP II) was examined using millisecond-phase transient-state kinetics. RAP74 deletion mutants RAP74(1-227), which includes an intact alpha1 helix, and RAP74(1-158), in which the alpha1 helix is deleted, were compared. Analysis of(More)
Although the quality of herbal medicine is tightly associated with plant genotype and location, microbial traits of most herbs remain unclear. In this study, bacterial communities residing Astragali Radix, which is derived from Astragalus membranaceus and A. mongholicus roots, have been characterized by automated ribosomal intergenic spacer analysis (ARISA)(More)
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