Baek, I. | Search bioRxiv

bioRxiv2021

Single molecule studies reveal branched pathways for activator-dependent pre-initiation complex assembly

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. HIGHLIGHTSSingle molecule microscopy reveals unexpected dynamics of RNA Pol II and GTFs Multiple Pol IIs cluster on UAS/enhancer-bound activators before binding the core promoter Pol II, TFIIF, and TFIIE, but not TFIIH, can pre-assemble at the UAS/enhancer Activators increase the rates of Pol II and GTF association with DNA eTOC BlurbSingle-molecule microscopy experiments by Baek et al. show that RNA polymerase II and basal transcription factors TFIIF and TFIIE preassemble on UAS/enhancer-bound activators, poised for loading into initiation complexes with TFIIH at the core promoter. Transcription activators kinetically enhance factor recruitment, creating a localized cluster of polymerases at the UAS/enhancer.

molecular biology↗

bioRxiv2020

Dynamics of RNA polymerase II and elongation factor Spt4/5 recruitment during activator-dependent transcription

In eukaryotes, RNA polymerase II (RNApII) transcribes messenger RNA from template DNA. Decades of experiments have identified the proteins needed for transcription activation, initiation complex assembly, and productive elongation. However, the dynamics of recruitment of these proteins to transcription complexes, and of the transitions between these steps, are poorly understood. We used multi-wavelength single-molecule fluorescence microscopy to directly image and quantitate these dynamics in a budding yeast nuclear extract that reconstitutes activator-dependent transcription in vitro. A strong activator (Gal4-VP16) greatly stimulated reversible binding of individual RNApII molecules to template DNA, with no detectable involvement of RNApII-containing condensates. Binding of labeled elongation factor Spt4/5 to DNA typically followed RNApII binding, was NTP-dependent, and was correlated with association of mRNA-binding protein Hek2, demonstrating specificity of Spt4/5 binding to elongation complexes. Quantitative kinetic modeling shows that only a fraction of RNApII binding events are productive and implies a rate-limiting step, probably associated with recruitment of general transcription factors, needed to assemble a transcription-competent pre-initiation complex at the promoter. Spt4/5 association with transcription complexes was slowly reversible, with DNA-bound RNApII molecules sometimes binding and releasing Spt4/5 multiple times. The average Spt4/5 residence time was of similar magnitude to the time required to transcribe an average length yeast gene. These dynamics suggest that a single Spt4/5 molecule remains associated during a typical transcription event, yet can dissociate from RNApII to allow disassembly of abnormally long-lived (i.e., stalled) elongation complexes. Significance StatementThe synthesis of a eukaryotic messenger RNA molecule involves the association of RNA polymerase and dozens of accessory proteins on DNA. We used differently colored fluorescent dyes to tag DNA, RNA polymerase II, and the elongation factor Spt4/5 in yeast nuclear extract, and then observed the assembly and dynamics of individual molecules of the proteins with single DNA molecules by microscopy. The observations quantitatively define an overall pathway by which transcription complexes form and evolve in response to an activator protein. They suggest how molecular complex dynamics may be tuned to optimize efficient RNA production.

biochemistry↗