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Edited by Taekjip Ha, Johns Hopkins University, Baltimore, MD, and approved November 4, 2020 (received for review June 1, 2020)
Significance
The 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 during activator-dependent transcription. They also suggest how Spt4/5 dynamics might promote efficient RNA production.
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.
Footnotes
↵1G.A.R. and I.B. contributed equally to this work.
- ↵2To whom correspondence may be addressed. Email: steveb{at}hms.harvard.edu or gelles{at}brandeis.edu.
Author contributions: G.A.R., I.B., L.J.F., Y.J.J., S.B., and J.G. designed research; G.A.R., I.B., L.J.F., Y.J.J., S.B., and J.G. performed research; G.A.R., I.B., L.J.F., Y.J.J., S.B., and J.G. analyzed data; and G.A.R., I.B., L.J.F., S.B., and J.G. wrote the paper.
The authors declare no competing interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2011224117/-/DCSupplemental.
Data Availability.
Source data for the single-molecule experiments are provided as “interval” files that can be read and manipulated by the Matlab program imscroll, which is publicly available in GitHub: https://github.com/gelles-brandeis/CoSMoS_Analysis. The source data are archived in Zenodo (DOI: 10.5281/zenodo.4065399).
Published under the PNAS license.
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Dynamics of RNA polymerase II and elongation factor Spt4/5 recruitment during activator-dependent transcription
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- Biological Sciences
- Biochemistry
- Physical Sciences
- Biophysics and Computational Biology
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