Single molecule microscopy reveals mechanistic insight into RNA polymerase II preinitiation complex assembly and transcriptional activity

Horn, Abigail E.; Kugel, Jennifer F.; Goodrich, James A.

doi:10.1093/nar/gkw321

Cited by 20 publications

(24 citation statements)

References 62 publications

(57 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We also caution that our preferred model is based on data from one synthetic promoter containing multiple canonical core promoter elements, whereas in vivo PIC assembly could use diverse pathways, depending on the composition of promoter structures and the local chromatin environment. Using a complementary single-molecule in vitro assay system, Goodrich and colleagues (Horn et al 2016) reported recently that TBP-TFIIB-TFIIF-Pol II can form a stable quadruple complex capable of supporting transcription, consistent with a partial preassembly model. In the future, simultaneous monitoring of multiple protein factors binding to immobilized promoter DNA, particularly when coupled to real-time nascent RNA detection , should help clarify how prevalent such hypothetical preassembled complexes may be and their functional relevance for promoter-specific gene regulation.…”

Section: Discussionmentioning

confidence: 76%

Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis

et al. 2016

View full text Add to dashboard Cite

Transcription of protein-encoding genes in eukaryotic cells requires the coordinated action of multiple general transcription factors (GTFs) and RNA polymerase II (Pol II). A "step-wise" preinitiation complex (PIC) assembly model has been suggested based on conventional ensemble biochemical measurements, in which protein factors bind stably to the promoter DNA sequentially to build a functional PIC. However, recent dynamic measurements in live cells suggest that transcription factors mostly interact with chromatin DNA rather transiently. To gain a clearer dynamic picture of PIC assembly, we established an integrated in vitro single-molecule transcription platform reconstituted from highly purified human transcription factors and complemented it by live-cell imaging. Here we performed real-time measurements of the hierarchal promoter-specific binding of TFIID, TFIIA, and TFIIB. Surprisingly, we found that while promoter binding of TFIID and TFIIA is stable, promoter binding by TFIIB is highly transient and dynamic (with an average residence time of 1.5 sec). Stable TFIIB-promoter association and progression beyond this apparent PIC assembly checkpoint control occurs only in the presence of Pol II-TFIIF. This transient-to-stable transition of TFIIB-binding dynamics has gone undetected previously and underscores the advantages of single-molecule assays for revealing the dynamic nature of complex biological reactions.

show abstract

Section: Discussionmentioning

confidence: 76%

Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis

et al. 2016

View full text Add to dashboard Cite

show abstract

“…This step renders the DNA competent (DNA*) for PIC and EC formation, and probably represents binding to the core promoter of a subset of the RNApII general transcription factors. In particular, it is known that at least TBP and TFIIB must be present before RNApII can proceed on the pathway to PIC formation (2,41). Alternative simpler mechanisms lacking the non-productive RNApII binding intermediate (DNA·RNAPII) did not produce satisfactory agreement with the data.…”

Section: Quantitative Kinetic Model For Initiation Complex Formationmentioning

confidence: 88%

“…3B), and therefore conclude that Spt4/5 binding is confined to ECs, consistent with our mass spectrometry observations. The kinetics of RNApII basal transcription initiation (i.e., initiation that occurs in the absence of activators) has been previously examined in vitro, including by single-molecule methods (41,42,(44)(45)(46). Such studies typically observe transcription from only a small fraction of template molecules, although this can be greatly improved by pre-formation of PICs before initiating RNA synthesis (47)(48)(49).…”

Section: Discussionmentioning

confidence: 99%

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

Rosen¹,

Baek

Friedman³

et al. 2020

Preprint

View full text Add to dashboard Cite

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 RNApIIcontaining 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.

show abstract

“…Recombinant human TBP, untagged TFIIB, TFIIF, and native human Pol II were prepared as described previously [20]. The ensemble in vitro transcription reactions were assembled as previously described [20]. Briefly, reactions were performed in 10% glycerol, Reactions were stopped and RNA transcripts were ethanol precipitated as described [16].…”

Section: Ensemble Transcription Assaysmentioning

confidence: 99%

Release of human TFIIB from actively transcribing complexes is triggered upon synthesis of 7 nt and 9 nt RNAs

Chen

Goodrich

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

AbstractRNA polymerase II (Pol II) and its general transcription factors assemble on the promoters of mRNA genes to form large macromolecular complexes that initiate transcription in a regulated manner. During early transcription these complexes undergo dynamic rearrangement and disassembly as Pol II moves away from the start site of transcription and transitions into elongation. One step in disassembly is the release of the general transcription factor TFIIB, although the mechanism of release and its relationship to the activity of transcribing Pol II is not understood. We developed a single molecule fluorescence transcription system to investigate TFIIB release in vitro. Leveraging our ability to distinguish active from inactive complexes, we found that nearly all transcriptionally active complexes release TFIIB during early transcription. Release is not dependent on the contacts TFIIB makes with its recognition element in promoter DNA. We identified two different points in early transcription at which release is triggered, reflecting heterogeneity across the population of actively transcribing complexes. TFIIB releases after both trigger points with similar kinetics, suggesting the rate of release is independent of the molecular transformations that prompt release. Together our data support the model that TFIIB release is important to maintain the transcriptional activity of Pol II as initiating complexes transition into elongation complexes.

show abstract

Single molecule microscopy reveals mechanistic insight into RNA polymerase II preinitiation complex assembly and transcriptional activity

Cited by 20 publications

References 62 publications

Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis

Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis

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

Release of human TFIIB from actively transcribing complexes is triggered upon synthesis of 7 nt and 9 nt RNAs

Contact Info

Product

Resources

About