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Review
. 2013 Dec;38(12):603-11.
doi: 10.1016/j.tibs.2013.09.002. Epub 2013 Oct 11.

Structural insights into transcription initiation by RNA polymerase II

Sebastian Grünberg  1 , Steven Hahn
Affiliations
Review

Structural insights into transcription initiation by RNA polymerase II

Sebastian Grünberg et al. Trends Biochem Sci. 2013 Dec.

Abstract

Transcriptional regulation is one of the most important steps in control of cell identity, growth, differentiation, and development. Many signaling pathways controlling these processes ultimately target the core transcription machinery that, for protein coding genes, consists of RNA polymerase II (Pol II) and the general transcription factors (GTFs). New studies on the structure and mechanism of the core assembly and how it interfaces with promoter DNA and coactivator complexes have given tremendous insight into early steps in the initiation process, genome-wide binding, and mechanisms conserved for all nuclear and archaeal Pols. Here, we review recent developments in dissecting the architecture of the Pol II core machinery with a focus on early and regulated steps in transcription initiation.

Keywords: RNA polymerase II; SAGA; TFIID; TFIIH; coactivators; mediator; open complex; preinitiation complex (PIC); transcription; translocase.

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Figures

Figure I
Figure I
Diagram of Ssl2 function in OC formation. In the left panel, upstream promoter DNA is in a fixed position due to binding by TBP-TFIIB-Pol II. Ssl2 lies on downstream DNA at a fixed position due to interaction with TFIIE. Ssl2 feeds DNA into the cleft by a right-handed threading mechanism, rotating and unwinding DNA. Figure reused with permission from [16].
Fig. 1
Fig. 1. Location of the general factors in the Pol II PIC
a) TFIIB domain organization. The domain color code (colored lettering) is used in panels a–c. b) Pol II-TFIIB complex [19]. TFIIB is shown as a cartoon, Pol II as a grey surface. Rpb2 is removed for clarity. The Rpb1 rudder (cyan) and clamp coiled-coil (light green) are highlighted. The TFIIB Zn-atom is shown as blue sphere, the two Mg-atoms in the Pol II active site are shown as pink spheres. c) TFIIB binding to Pol II results in ordering of the Rpb2 protrusion. To visualize the protrusion (light purple), Rpb2 (partially transparent) has been added and Pol II has been rotated upwards by ~40 degrees compared to panel 1b. d) TFIIF binding to the PIC. As positioned by site-specific protein cleavage and crosslinking assays [13, 15], dimerization domains of TFIIF subunits Tfg1 (deep purple) and Tfg2 (blue) are located on the Pol II lobe. A C-terminal winged helix (WH) domain in Tfg2 resides on top of the Pol II protrusion domain, close to upstream promoter DNA. In contrast, a cryo EM structure suggested that the human TFIIF WH lies just downstream from the TFIIB core domain (not shown). TFIIB is shown in yellow, TBP in green. EM showed that promoter DNA is slightly bent over the cleft by ~ 18 degrees and the Pol II clamp is in an open state to accommodate dsDNA for the initial step in OC formation [21]. e) A triple WH structure of TFIIE spans the Pol II cleft. As positioned by site-specific protein cleavage assays [16], Tfa1 WH domain (cyan) anchors TFIIE on the Pol II clamp and dimerizes with the Tfa2 tandem WH domain (red) that encircles promoter DNA at positions 18 to 8 relative to the human TSS at +1.
Fig. 2
Fig. 2. Structure of TFIIH
The modules containing the cyclin-dependent kinase (CDK)-activating kinase complex (CAK), Ssl2/XPB, and Rad3/XPD are labeled. a) Negative stain EM image of yeast TFIIH [41]. The locations of subunits Ssl2, Rad3 and the CAK module (identified by EM analysis of TFIIH lacking these subunits or by gold labeling) are indicated. b) Negative stain EM image of human TFIIH [21]. c) Alignment of both densities, showing yTFIIH in grey and hTFIIH as pink mesh. EM data suggest a similar general TFIIH shape with several significant differences.
Fig. 3
Fig. 3. Architecture of the basal PIC
Components of the PIC are colored according to the color scheme shown in the color chart. 3D coordinates of the model and a movie are available as supplemental material. a) PIC model based on crystal structures and biochemical mapping. Note that the two Ssl2 RecA-like domains (labeled Ssl2 C-term and Ssl2 N-term) abut TFIIE, encircling promoter DNA at positions −2 to +6 regarding the human transcription start site at +1. b) Negative stain structure of the human PIC, containing all GTFs and Pol II [21]. EM density corresponding to the TFIIH core complex is shown in brown. Crystal structures and homology model of PIC components were docked into the EM density, suggesting Ssl2 binding to downstream promoter DNA at positions +10 to +20. Note that models in panels a and b show different downstream locations for Ssl2, either adjacent to TFIIE or in a more downstream location.
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