Mechanistic studies of RNAPII initiation, re-initiation and bursting

The Pre-Initiation Complex (PIC), which consists of Mediator, TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and RNA polymerase II (RNAPII), governs the expression of all protein-coding and most non-coding RNA genes in the human genome and is over 4.0 MDa in size. Mediator and TFIID, each approximately 1.4 MDa, represent major assemblies within the PIC and work to control PIC function in ways that remain poorly understood. The structural features of Mediator and TFIID (large cavities, extended shape) coupled with their large size (e.g. Mediator approximately 320 x 180 x 160 Å) provide an extensive surface for interaction with other protein complexes within the PIC. TFIIH is also a large (10 subunits, 0.5MDa) protein complex. Like Mediator and TFIID, TFIIH is required for RNAPII transcription, genome-wide. Notably, Mediator, TFIID, and TFIIH appear to function cooperatively within the PIC; however, the molecular mechanisms underlying their coordinated or synergistic function remain poorly understood. It also remains unclear how "post-initiation" regulatory factors such as P-TEFb, NELF, DSIF, or the Mediator kinase module govern RNAPII promoter-proximal transcription or re-initiation/bursting. Our lab has identified key roles for TFIID, Mediator, and the TFIIH-associated kinase CDK7 in the regulation of RNAPII initiation, promoter-proximal pausing, and pause release, and we continue to work in this high-impact area.

PausePIC scheme

Although the basic mechanisms underlying RNAPII initiation are fairly well studied, re-initiation mechanisms, which involve rapid engagement of a second (or third) RNAPII enzyme, remain poorly understood. Re-initiation is related to transcriptional bursting, which is generally defined as periods of activity followed by periods of inactivity. Notably, all RNAPII-transcribed genes in human cells undergo bursts of expression, punctuated by periods of inactivity. Our lab is using a variety of biochemical, cellular, and biophysical methods to better define how Mediator, TFIID, and TFIIH work together with TFs and other PIC components to regulate RNAPII re-initiation and bursting. For example, we used a reconstituted human PIC system and smTIRF microscopy to demonstrate that TFs and Mediator act synergistically to drive RNAPII bursting (Palacio & Taatjes bioRxiv 2025 631569). We are continuing our work on re-initiation and bursting, with a goal of defining the molecular mechanisms required for this fundamentally important, enigmatic process. 

RIFT re-init

Recent publications related to this topic:

Palacio M; Taatjes DJ. Real-time visualization of reconstituted transcription reveals RNA polymerase II activation mechanisms at single promoters. bioRxiv 2025, 631569.

Luyties, O; Sanford, L; Rodino, J; Nagel, M; Jones, T; Rimel, JK; Ebmeier, CC; Palacio, M; Shelby, GS; Cozzolino, K; Brennan, F; Hartzog, A; Saucedo, MB; Watts, LP; Spencer, S; Kugel, JF; Dowell, RD; Taatjes DJ. Multi-omics and biochemical reconstitution reveal CDK7-dependent mechanisms controlling RNA polymerase II function at gene 5'- and 3'-ends. Cell Rep 2025, 44: 115904.

Nagel, M; Taatjes, DJ. Regulation of RNA polymerase II transcription through re-initiation and bursting. Mol Cell 2025, 85: 1907-1919.

Jones, T; Feng, J; Luyties, O; Cozzolino, K; Sanford, L; Rimel, JK; Ebmeier, CC; Shelby, GS; Watts, LP; Rodino, J; Rajagopal, N; Hu, S; Brennan, F; Maas, ZL; Alnemy, S; Richter, WF; Koh, AF; Cronin, NB; Madduri, A; Das, J; Cooper, E; Hamman, KB; Carulli, JP; Allen, MA; Spencer, S; Kotecha, A; Marineau, J; Greber, BJ; Dowell, RD; Taatjes DJ. TFIIH kinase CDK7 drives cell proliferation through a common core transcription factor network. Sci Adv 2025, 11: eadr9660. 

Richter, WF; Taatjes DJ. Changing structures, changing paradigms: NELF helps regulate paused or elongating RNA Polymerase II. Mol Cell 2024, 84: 1180 - 1182.  

Nayak, S; Taatjes DJ. SnapShot: Mediator Complex Structure. Cell 2022, 185: 3458.

Bassett, J; Rimel, JK; Basu, S; Basnet, P; Luo, J; Engel, KL; Nagel, MT; Woyciehowsky, A; Ebmeier, CC; Kaplan, CD; Taatjes, DJ; Ranish, JA. Systematic mutagenesis of TFIIH subunit p52/Tfb2 identifies residues required for XPB/Ssl2 subunit function and genetic interactions with TFB6. J Biol Chem 2022, 102433.

Richter, WF; Nayak, S; Iwasa, J; Taatjes, DJ. The Mediator complex as a master regulator of transcription by RNA polymerase II. Nat Rev Mol Cell Biol 2022, 23: 732 - 749.

Luyties, O; Taatjes, DJ.  The Mediator kinase module: an interface between cell signaling and transcription. Trends Biochem Sci 2022, 47: 314 - 327.  

Schier, A; Taatjes, DJ. Everything at once: Cryo-EM yields remarkable insights about human RNA polymerase II transcription. Nat Struct Mol Biol 2021, 28: 540 - 543.  

Tomko, EJ; Luyties, O; Rimel, JK; Tsai, C; Fuss, JO; Fishburn, J; Hahn, S; Tsutakawa, SE; Taatjes, DJ; Galburt, EA. The role of XPB/Ssl2 dsDNA translocation processivity in transcription start-site scanning. J Mol Biol 2021; 433: 166813.

Rimel, JK; Poss, ZC; Erickson, B; Maas, ZL; Ebmeier, CC; Johnson, JL; Decker, T-M; Yaron, TM; Bradley, MJ; Hamman, KB; Hu, S; Malojcic, G; Marineau, JJ; White, PW; Brault, M.; Tao, L.; DeRoy, P; Clavette, C; Nayak, S; Damon, LJ; Kaltheuner, IH; Bunch, H; Cantley, LC; Geyer, M; Iwasa, J; Dowell, RD; Bentley, DL; Old WM; Taatjes, DJ.  Selective inhibition of CDK7 reveals high-confidence targets and novel mechanisms for TFIIH function in transcription. Genes Dev 2020; 34: 1452 – 1473.

Fant, CB; Levandowski, CB; Gupta, K; Maas, ZL; Moir, JT; Rubin, JD; Sawyer, A; Esbin, M; Rimel, JK; Luyties, O; Marr, MT; Berger, I; Dowell, RD; Taatjes, DJ. TFIID enables RNA polymerase II promoter-proximal pausing. Mol Cell 2020, 78: 785 – 793. 

Schier, AC; Taatjes, DJ. Structure and mechanism of the RNA polymerase II transcription machinery. Genes Dev. 2020, 34: 465 – 488. 

Guo, YE; Manteiga, JC; Henninger, J; Sabari, BR; Dall'Agnese, A; Hannett, NM; Spille, J-H; Afeyan, LK; Zamudio, AV;  Shrinivas, K; Abraham, BJ; Boija, A; Decker, TM; Rimel, JK; Fant, CB; Lee, TI; Cisse, II; Sharp, PA; Taatjes, DJ; Young, RA. RNA polymerase II phosphorylation regulates a switch between transcriptional and splicing condensates. Nature 2019, 572: 543 – 548.

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