Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Review
. 2020 Apr 1;34(7-8):465-488.
doi: 10.1101/gad.335679.119.

Structure and mechanism of the RNA polymerase II transcription machinery

Allison C Schier  1 , Dylan J Taatjes  1
Affiliations
Review

Structure and mechanism of the RNA polymerase II transcription machinery

Allison C Schier et al. Genes Dev. .

Abstract

RNA polymerase II (Pol II) transcribes all protein-coding genes and many noncoding RNAs in eukaryotic genomes. Although Pol II is a complex, 12-subunit enzyme, it lacks the ability to initiate transcription and cannot consistently transcribe through long DNA sequences. To execute these essential functions, an array of proteins and protein complexes interact with Pol II to regulate its activity. In this review, we detail the structure and mechanism of over a dozen factors that govern Pol II initiation (e.g., TFIID, TFIIH, and Mediator), pausing, and elongation (e.g., DSIF, NELF, PAF, and P-TEFb). The structural basis for Pol II transcription regulation has advanced rapidly in the past decade, largely due to technological innovations in cryoelectron microscopy. Here, we summarize a wealth of structural and functional data that have enabled a deeper understanding of Pol II transcription mechanisms; we also highlight mechanistic questions that remain unanswered or controversial.

Keywords: DSIF; Mediator; NELF; P-TEFb; TBP; TFIID; TFIIH; cryo-EM; pausing; preinitiation complex.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Overview of the PIC and DNA path in closed and open complex. (A) The preinitiation complex (PIC) consists of TFIIA (red), TFIIB (orange), TFIID (not pictured), TFIIE (cyan), TFIIF (magenta), TFIIH (maroon), RNA polymerase II (Pol II, gray), and promoter DNA (blue). Upstream promoter DNA is bound by TFIIB, TBP, TFIIA, and TFIIF. Downstream DNA is bound by TFIIH and TFIID. After promoter opening, TFIIB, TFIIE, and TFIIF interact with and stabilize the ssDNA in the Pol II cleft. PIC is shown with TFIIH (left) and without TFIIH (right). Adapted from PDB 5IY7 (He et al. 2016). (B) Promoter DNA before and after opening. Closed complex (CC) DNA is mostly linear with the characteristic 90° bend at the TATA box (purple). Upon promoter opening, the strands separate at the transcription start site (denoted Inr, light pink). Center and right images show Pol II overlaid with the DNA to show location in the active site. The center view shows the top of Pol II, with ssDNA extending into the active site. The right view shows the front of Pol II with the DNA. Adapted from PDB 5IY6 and PDB 5IY7 (He et al. 2016).
Figure 2.
Figure 2.
TBP is a regulatory hub within the larger TFIID complex. (A) Structure of TBP (yellow), TFIIA (red), and TFIIB (orange) bound to promoter DNA in an open PIC complex. The TFIIB zinc ribbon (amino acids 1–57, amino acids 7–57 pictured) is important for recruiting Pol II to the TSS. Amino acids 56–60 in the TFIIB zinc ribbon and B-reader (amino acids 58–84) domains stabilize the open template strand, whereas amino acids 86, 98–100, and 103 in the linker domain (amino acids 85–123) interact with the nontemplate strand (Kostrewa et al. 2009). TFIIA residues important for TBP interaction (TBP residues 187–208): 345–349, 375, and 376 from TFIIA subunit 1, and 65–67 in TFIIA subunit 2. TFIIB residues that interact with TBP (residues 271, 274, 278, 283–287, 306, and 337): 169, 177, 188, 195, 205, 208, 243, 246, 247, and 249. Adapted from PDB 5IY7 (He et al. 2016). (B) MOT1 (light green) binds TBP (yellow) and displaces in an ATP-dependent manner. MOT1 contains 16 HEAT repeats that bind multiple regions of TBP. MOT1 also binds upstream DNA; MOT1 contains a “latch” (amino acids 94–132) that blocks TBP–DNA reassociation. Adapted from PDB 3OC3 (Wollmann et al. 2011). (C) NC2 (pink) binds TBP (yellow) and DNA to negatively regulate transcription. NC2 binds the DNA major groove and an NC2 α helix (amino acids 180–210) sterically blocks TFIIB–TBP interactions. Adapted from PDB 1JFI (Kamada et al. 2001). (D) Overall structure of human TFIIB in the PIC. Structural domains include the TFIIB core (with two cyclin folds), the linker (amino acids 85–123), the reader (amino acids 58–84), and the zinc ribbon (amino acids 1–57). These domains are important for stabilizing single-stranded promoter DNA in the open complex. Adapted from PDB 5IY7 (He et al. 2016). (E) Structure of TFIID bound to promoter DNA, along with TFIIA and TBP. TFIIA–TFIIB–TBP bind upstream of the TSS. In this example, the supercore promoter (SCP) was used (Juven-Gershon et al. 2006), which has multiple promoter elements, some or all of which are not found in promoters genome-wide. However, the protein–DNA interactions shown here are likely to occur at promoters with different sequences. SCP upstream DNA elements are the BREu (−37 to −32), TATA (−31 to −24), and BREd (−23 to −17). TFIIA residues 68–71 and 35–27 interact with the TAF4/12 dimer in TFIID lobe B (TAF4 residues 1002-1007; TAF12 residue 75). TAF1 residues 972, 996, 1022, and 1023 bind the Inr sequence (−3 to +3, with +1 shown in light blue); residues 797, 843, 844, 852, and 862 bind the motif ten element (MTE; +18 to +27), and TAF1 residues 839, 843, 852, 856, and 858 bind the downstream promoter element (DPE; +28 to +34). TAF2 (residue 543) also interacts with the MTE. Another interaction involves the TAF4 hairpin (amino acids 966–1000) and upstream DNA at the end of the BREd. Adapted from PDB 5IY7 (He et al. 2016) and PDB 6MZM (Patel et al. 2018). (F) The structure in E includes only structured domains; here, we show a rendering of the “rearranged” free TFIID structure (semiopaque), which shows the entire TFIID density (i.e., including disordered regions). The free TFIID structure (adapted from EMD-2284) (Cianfrocco et al. 2013) was visually aligned and superimposed onto the structure shown in E. This rendering highlights additional TFIID density downstream from the MTE/DPE promoter elements and shows an approximate position of the mobile lobe A, behind lobe B. Adapted from PDB 6MZM (Patel et al. 2018). Amino acid interactions were determined using the “find any contacts” function in PyMol, set to 4 Å. Amino acids listed correspond to the human proteins.
Figure 3.
Figure 3.
Pol II and TFIIF. (A) Bovine Pol II (gray) shown in two orientations (rotated 180°). DNA is colored in blue, and the Pol II stalk, clamp, foot, funnel, and RNA exit channel are marked; the protrusion and foot domains are shown in dark gray. The inset shows a zoomed-in view of the Pol II active site, with the trigger loop (Rpb1 amino acids 1095–1130) shown in red, bridge helix (Rpb1 amino acids 833–869) in cyan, rudder (Rpb1 amino acids 318–338) in yellow, fork loop 1 (Rpb2 amino acids 461–480) in green, and fork loop 2 (Rpb2 amino acids 499–520) in magenta (Cramer et al. 2001). Adapted from PDB 5FLM (Bernecky et al. 2016). (B) Structure of TFIIF in the PIC. RAP30 (TFIIFβ) is shown in light pink, and RAP74 (TFIIFα) is shown in light pink. (C) The same view of TFIIF is shown, with open promoter DNA and TBP added. The RAP30 winged helix (WH) domain (amino acids 181–240) interacts with the upstream DNA (−37 to −32) to help stabilize promoter DNA. The RAP30 linker (amino acids 119–175) interacts with TBP and may aid in positioning the WH domain. TBP residues 194 and 195 are implicated in the interaction, with RAP30 residues 172, 174, and 176. Adapted from PDB 5IY7 (He et al. 2016). Amino acid interactions were determined using the “find any contacts” function in PyMol, set to 4 Å. Amino acid residues listed correspond to bovine (panel A) or human proteins.
Figure 4.
Figure 4.
Structural details for TFIIE and TFIIH. (A) TFIIE and TFIIH converge at the Pol II stalk. The Ring domain in the TFIIH subunit MAT1 interacts with the OB domain of RPB7 and the TFIIE linker helices. Amino acids involved in the interactions are RPB7 164–168 and MAT1 40–45, 55, and 56. RPB7 also interacts with TFIIE, through residues 91–96, 105–107, 111, 151, 153, 158, and 160 (RPB7) and 124, 137–143, 145, 150–152, 161, and 162 (TFIIEα). PyMol was unable to detect any contacts within 4 Å between the IIE linker helix and the MAT1 ring domain in the human PIC, but they were identified in the yeast PIC (Schilbach et al. 2017). Adapted from PDB 5IY7 (He et al. 2016). (B) The structure of the free human TFIIH core complex, with MAT1. MAT1 helps link the two ATPase subunits XPB and XPD. MAT1 is in blue, XPB is in purple, XPD is in red, p8 is in green, p62 is in cyan, p34 is in magenta, and p44 is in orange. Not shown are the kinase module subunits CDK7 and CCNH. Adapted from PDB 6NMI (Greber et al. 2019). (C) Additional detail for MAT1 interactions with XPB and XPD. The XPD–MAT1 interaction involves over a dozen residues between 250 and 370 (XPD), plus residues 641 and 642, and about a dozen residues between 1 and 161 in MAT1. The XPB–MAT1 interaction involves residues 174, 183, 186, 189, 190, 195, 198, and 200–203 (XPB) and MAT1 residues 174, 177, 181, 184, 185, 188, and 194. Adapted from PDB 6NMI (Greber et al. 2019). (D) The DNA repair protein XPA displaces MAT1 at its XPB and XPD interfaces, and causes structural changes in XPD and XPB. XPA binding also allows rearrangement of core TFIIH subunits to fully engage the translocase and helicase functions of the complex (Kokic et al. 2019). Key residues involved in the interaction are 421, 422, 425, 714, 718, and 720 (XPB) and XPA residues 153, 157–159, 232, and 235–237; residues 634, 638, 641, 645, 647, and 648 (XPD) and XPA residues 164–166, 168, 174, 177, and 179. Adapted from PDB 6RO4 (Kokic et al. 2019). Amino acid interactions were determined using the “find any contacts” function in PyMol, set to 4 Å. Amino acids listed correspond to the human proteins.
Figure 5.
Figure 5.
PIC structural models that include the Mediator complex. (A) Model of a partial human PIC that includes Mediator. Figure was prepared by rendering a Mediator–Pol II–TFIIF cryoEM density (Bernecky et al. 2011) in PyMol as a semiopaque black mesh, then visually aligned to a human PIC structure (He et al. 2016). Colors for each PIC factor are identical to Figure 1. The top row shows two views of the complex without TFIIH, and the bottom row shows the same views with TFIIH. Structural remodeling is likely upon binding TFIIH, as clashes are evident in this artificially docked model. The differences between yeast (see B) and human Mediator reflect the much larger size of the human Mediator complex (Table 1). However, the orientation of the human Mediator complex modeled in the human PIC is distinct from the yeast PIC. These differences could result from true differences in PIC structure (yeast vs. human) or could simply result from the fact that the human PIC model is not derived from a single complete structural assembly, as done for the yeast PIC (Schilbach et al. 2017). Adapted from PDB 5IY7 (He et al. 2016) and EMD-5343 (Bernecky et al. 2011). (B) Structure of a yeast PIC (S. cerevisiae), shown in identical orientations with A, based on alignment of Pol II. Here, a core Mediator complex is shown in green, whereas all other PIC factors are shown in the same colors as A. The top row shows two views of the complex without TFIIH, and the bottom row shows the same views with TFIIH. The different orientation of downstream DNA (vs. A) reflects potential structural differences between yeast and human PICs. Note, however, that A is a hypothetical model that merges two different structures, whereas B represents cryoEM data from a single structure (Schilbach et al. 2017). Adapted from PDB 5oqm (Schilbach et al. 2017).
Figure 6.
Figure 6.
Pol II elongation complexes bound to pausing (NELF) or elongation factors (DSIF, PAF, SPT6). (A) Structures of partial PICs that emphasize how DSIF binds Pol II surfaces occupied by TFIIB, TFIIE, and TFIIF. At left is a Pol II structure with TBP, TFIIB, TFIIE, and TFIIF. At right is a Pol II structure bound to DSIF and NELF. Adapted from PDB 6GML (Vos et al. 2018b) and PDB 5IY7 (He et al. 2016). (B) Structure of TFIIS bound to Pol II. Pol II is shown in gray and TFIIS is shown in green; the Rpb1 jaw is shown in red, the Rpb1 funnel is shown in cyan, and Rpb5 is shown in orange. TFIIS amino acids 230–301 extend into the Pol II funnel, which positions TFIIS residues D290 and E291 near a catalytic zinc ion, which helps catalyze cleavage of backtracked RNA. Adapted from PDB 5IY7 (He et al. 2016). (C) Structure of NELF and DSIF bound to a transcribing/paused Pol II. DNA is shown in blue, nascent RNA is shown in salmon, NELF is shown in teal, SPT5 is shown in yellow, and SPT4 is shown in olive. Select NELF/DSIF domains or subunits are shown, with two views rotated 180°. Adapted from PDB 6GML (Vos et al. 2018b). (D) Detail from the DSIF/NELF–Pol II structure, showing the interaction between NELFC and the Pol II trigger loop (RPB1 amino acids 1095–1130). Adapted from PDB 6GML (Vos et al. 2018b). (E) Two views (rotated 180°) of the PAF complex, SPT6, and DSIF bound to Pol II. Pol II is shown in gray, DSIF is shown in yellow, SPT4 is shown in olive, DNA is shown in blue, RNA is shown in salmon, PAF is shown in purple, and SPT6 is shown in neon green. Adapted from PDB 6GMH (Vos et al. 2018a). (F) NELF–Pol II binding is mutually exclusive with PAF–Pol II binding. The NELF–DSIF–Pol II structure is shown in the same orientation as the right-hand image in E. The WRD61 and CTR9 subunits of PAF directly clash with NELFA/C, NELFB/C, and NELFB/E. Adapted from PDB 6GML (Vos et al. 2018b).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Abdulrahman W, Iltis I, Radu L, Braun C, Maglott-Roth A, Giraudon C, Egly JM, Poterszman A. 2013. ARCH domain of XPD, an anchoring platform for CAK that conditions TFIIH DNA repair and transcription activities. Proc Natl Acad Sci 110: E633–E642. 10.1073/pnas.1213981110 - DOI - PMC - PubMed
    1. Adelman K, Marr MT, Werner J, Saunders A, Ni Z, Andrulis ED, Lis JT. 2005. Efficient release from promoter-proximal stall sites requires transcript cleavage factor TFIIS. Mol Cell 17: 103–112. 10.1016/j.molcel.2004.11.028 - DOI - PubMed
    1. Alekseev S, Nagy Z, Sandoz J, Weiss A, Egly JM, Le May N, Coin F. 2017. Transcription without XPB establishes a unified helicase-independent mechanism of promoter opening in eukaryotic gene expression. Mol Cell 65: 504–514.e4. 10.1016/j.molcel.2017.01.012 - DOI - PubMed
    1. Anandapadamanaban M, Andresen C, Helander S, Ohyama Y, Siponen MI, Lundström P, Kokubo T, Ikura M, Moche M, Sunnerhagen M. 2013. High-resolution structure of TBP with TAF1 reveals anchoring patterns in transcriptional regulation. Nat Struct Mol Biol 20: 1008–1014. 10.1038/nsmb.2611 - DOI - PMC - PubMed
    1. Andel F 3rd, Ladurner AG, Inouye C, Tjian R, Nogales E. 1999. Three-dimensional structure of the human TFIID–IIA–IIB complex. Science 286: 2153–2156. 10.1126/science.286.5447.2153 - DOI - PubMed

Publication types

Substances

-