Journal
MOLECULAR CELL
Volume 81, Issue 17, Pages 3576-+Publisher
CELL PRESS
DOI: 10.1016/j.molcel.2021.07.025
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Funding
- NIH [R01GM046498, R01GM081648, R01CA246500]
- Harvard Medical School
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Research shows that in activator-dependent PIC assembly, RNA Pol II, TFIIF, and TFIIE can preassemble on enhancer-bound activators, and multiple RNA Pol II complexes can simultaneously bind to form a localized cluster, while TFIIH binding is unique and depends on the basal promoter.
RNA polymerase II (RNA Pol II) transcription reconstituted from purified factors suggests pre-initiation complexes (PICs) can assemble by sequential incorporation of factors at the TATA box. However, these basal transcription reactions are generally independent of activators and co-activators. To study PIC assembly under more realistic conditions, we used single-molecule microscopy to visualize factor dynamics during activator-dependent reactions in nuclear extracts. Surprisingly, RNA Pol II, TFIIF, and TFIIE can preassemble on enhancer-bound activators before loading into PICs, and multiple RNA Pol II complexes can bind simultaneously to create a localized cluster. Unlike TFIIF and TFIIE, TFIIH binding is singular and dependent on the basal promoter. Activator-tethered factors exhibit dwell times on the order of seconds. In contrast, PICs can persist on the order of minutes in the absence of nucleotide triphosphates, although TFIIE remains unexpectedly dynamic even after TFIIH incorporation. Our kinetic measurements lead to a new branched model for activator-dependent PIC assembly.
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