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

Abstract

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 multiwavelength 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. 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 preinitiation 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.

Single molecule studies reveal branched pathways for activator-dependent assembly of RNA polymerase II pre-initiation complexes

“…2C, top, red dashed lines and Table S4), with initial specific binding of Pol II to DNA behaving as a single rate-limiting process with an apparent first-order association rate constant, (3.8 ± 1.0) × 10 -3 s -1 (Fig. 2E, left bar graph, blue), similar to that measured in the presence of NTPs (Rosen et al, 2020). DNA-specific Pol II binding was completely activator-dependent, as control experiments lacking Gal4-VP16 showed no DNA binding above the non-specific background (Fig.…”

“…2A. As previously observed in the presence of NTPs (Rosen et al, 2020), binding events of various durations were recorded. Individual DNAs often exhibited serial Pol II binding and dissociation events (Fig.…”

“…Yeast nuclear extracts were prepared as previously described (Rosen et al, 2020;Sikorski et al, 2012). Briefly, yeast cells were grown in 4 liters of YPD (3% dextrose) medium at 30°C to an OD600 of 3-4 and were harvested.…”

“…In our earlier study, we showed that overall Pol II binding to DNA behaved as a single rate-limiting step, yet there was a time lag before binding of the subset of Pol II molecules that proceeded on to elongation. Kinetic modeling suggested the existence of an additional slow step needed to allow Pol II binding to progress to PIC and elongation complexes (Rosen et al, 2020). We postulated that Pol II might initially bind Gal4-VP16 at the UAS, with arrival of TBP or other GTFs at the core promoter being rate-limiting for subsequent Pol II incorporation into the PIC.…”

“…In vivo single-molecule imaging has therefore emerged as an essential complementary approach for studying factor dynamics, but comes with its own limitations (Zhang and Tjian, 2018). To bridge this gap, we developed a system for fluorescently labeling and visualizing single transcription factor molecules within yeast nuclear extract (Rosen et al, 2020). Containing the full repertoire of nuclear factors, these extracts approximate physiological conditions better than reactions reconstituted with a limited set of purified factors.…”