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Dynamics of RNA polymerase II and elongation factor Spt4/5 recruitment during activator-dependent transcription
Tuesday, 2020/12/29 | 08:39:58
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Grace A. Rosen, Inwha Baek, Larry J. Friedman, Yoo Jin Joo, Stephen Buratowski, and Jeff Gelles PNAS December 22, 2020 117 (51) 32348-32357 SignificanceThe 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. AbstractIn 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.
See https://www.pnas.org/content/117/51/32348
Figure 1: Detection of RNApII and Spt4/5 binding to individual surface-tethered DNA488 molecules in Rpb1SNAP549/Spt5DHFR-Cy5 S. cerevisiae nuclear extract. (A) Schematic of DNA488 transcription template. This DNA contains five upstream Gal4 binding sites (yellow) and the CYC1 core promoter (green) with its transcription start site (bent arrow), followed by a 300-bp cassette (pink) encoding a G-less RNA. The template has attached biotin and AF488 dye (blue star) moieties. (B) Experimental scheme. DNA488 molecules immobilized on the surface of a flow chamber (blue) were at time t = 0 incubated with yeast nuclear extract containing dye (stars)-labeled proteins Rpb1SNAP549 and Spt5DHFR-Cy5 along with unlabeled general transcription factors (GTFs) and other nuclear proteins. Reactions were supplemented with recombinant Gal4-VP16 activator. RNApII and Spt4/5 binding to DNA were detected as colocalization of spots of green- and red-excited fluorescence at locations of blue-excited DNA spots. (C) Images of the same microscope field of view (65 × 65 µm) in the red-, green-, and blue-excited fluorescence channels taken at various times before (blue) and after (red and green) extract addition at time t = 0. Insets show magnified views of the marked region. Absence or presence of a spot of fluorescence colocalized with a particular DNA molecule are shown by open and filled arrowheads, respectively. |
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