PT  - JOURNAL ARTICLE
AU  - Baek, Inwha
AU  - Friedman, Larry J.
AU  - Gelles, Jeff
AU  - Buratowski, Stephen
TI  - Single molecule studies reveal branched pathways for activator-dependent pre-initiation complex assembly
AID  - 10.1101/2021.06.20.449130
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.06.20.449130
4099  - http://biorxiv.org/content/early/2021/06/20/2021.06.20.449130.short
4100  - http://biorxiv.org/content/early/2021/06/20/2021.06.20.449130.full
AB  - RNA polymerase II (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, Pol II, TFIIF, and TFIIE can pre-assemble on enhancer-bound activators before loading into PICs, and multiple 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.HIGHLIGHTS Single molecule microscopy reveals unexpected dynamics of RNA Pol II and GTFsMultiple Pol IIs cluster on UAS/enhancer-bound activators before binding the core promoterPol II, TFIIF, and TFIIE, but not TFIIH, can pre-assemble at the UAS/enhancerActivators increase the rates of Pol II and GTFs association with DNACompeting Interest StatementThe authors have declared no competing interest.