Structure of an RNA polymerase II preinitiation complex

Abstract

The structure of a 33-protein, 1.5-MDa RNA polymerase II preinitiation complex (PIC) was determined by cryo-EM and image processing at a resolution of 6-11 Å. Atomic structures of over 50% of the mass were fitted into the electron density map in a manner consistent with protein-protein cross-links previously identified by mass spectrometry. The resulting model of the PIC confirmed the main conclusions from previous cryo-EM at lower resolution, including the association of promoter DNA only with general transcription factors and not with the polymerase. Electron density due to DNA was identifiable by the grooves of the double helix and exhibited sharp bends at points downstream of the TATA box, with an important consequence: The DNA at the downstream end coincides with the DNA in a transcribing polymerase. The structure of the PIC is therefore conducive to promoter melting, start-site scanning, and the initiation of transcription.-E, -F, and -H, are required for the initiation of RNA polymerase II (pol II) transcription. The GTFs can be assembled with pol II and promoter DNA in a preinitiation complex (PIC) capable of efficient conversion to a transcribing complex (0.1-0.3 transcripts per template). Cryo-EM of the PIC at a resolution of 15-20 Å revealed a bipartite structure, with a P-lobe containing pol II and a G-lobe containing most of the mass of the GTFs (1). A cylinder of electron density, attributed to promoter DNA, was in contact only with GTFs and not with pol II. This architecture was consistent with the pathway of assembly of the PIC, beginning with a complex of promoter DNA and all but one of the GTFs, to which a complex of pol II and the remaining GTF was added (2).A combination of chemical cross-linking and mass spectrometry was used to locate the subunits of the GTFs in the low-resolution cryo-EM electron density map of the PIC. TFIIB was in proximity to promoter DNA at the upstream end of the pol II active center cleft, whereas Ssl2, a subunit of TFIIH, was in apparent contact with promoter DNA at the downstream end of the cleft. TFIIB bridges between pol II and promoter DNA, whereas Ssl2 is a DNA helicase, responsible for unwinding promoter DNA.The association of promoter DNA with the GTFs and not with pol II may be viewed as a fundamental principle of the PIC. Doublestranded DNA is straight and relatively rigid, whereas DNA would need to bend by about 90°to bind in the pol II active center cleft. The GTFs assemble on the double-stranded DNA, position it above the active center, and unwind the DNA. The resulting singlestranded region is flexible and can bend and bind in the pol II cleft.Cryo-EM of the PIC with state-of-the-art instrumentation has resulted in an electron density map at higher resolution. The improved map has confirmed and extended the previous findings and conclusions. ResultsCryo-EM and 3D Reconstruction. A PIC was formed as described (2) on an 86-bp fragment of HIS4 promoter DNA fragment, with pol II, GTFs (TFIIA, TFIIB, TBP, TFIIE, TFIIF, and holo-TFIIH), TFIIS, and with the ad...

Conserved architecture of the core RNA polymerase II initiation complex

“…Finally, our core ITC model can explain the XL-MS data obtained recently with a complete yeast PIC 8 . In the latter study, 117 distance restraints were obtained for Pol II, TFIIB, TFIIF and TBP.…”

“…The position of the Tfg2 WH domain above the cleft apparently represents its position in a complete ITC. This position is near TFIIE and TFIIA in the PIC 7,8 , and is likely stabilized on TFIIE and/or TFIIA binding. The other WH domain in TFIIF subunit Tfg1 only gave rise to a single crosslink at the Pol II jaw, and does not adopt a defined location 12 .…”

“…The architecture of the human PIC was obtained by electron microscopy (EM) 7 , and generally resembled that of the yeast PIC. Recently, an alternative model of the yeast PIC was derived based on a combination of EM and protein crosslinking coupled to mass spectrometry (XL-MS) 8 , raising the question whether the PIC architecture is indeed conserved between eukaryotic species.…”

Structural insights into transcription initiation by yeast RNA polymerase I

“…Surprisingly, in the Pol I PIC, the upstream DNA duplex follows a remarkably different path (Fig 2C). In the Pol II PIC, the upstream DNA is bound outside of the cleft by several transcription factors (Murakami et al , 2015; He et al , 2016; Plaschka et al , 2016), whereas in the Pol I PIC, the upstream DNA is positioned closer to the wall and protrusion domains. Moreover, upstream DNA in the Pol I elongation complex (EC) re‐anneals close to the protrusion domain and the positive helix (Tafur et al , 2016), while in the Pol I PIC, the upstream DNA is even closer to the wall and to the A135 “wedge” (residues 813–819; Barnes et al , 2015; Fig EV2A).…”

“…Furthermore, the C‐terminal cyclin domain of Rrn7 is not positioned as closely to the DNA as in Pol II, where the C‐terminal cyclin domain of TFIIB contacts DNA upstream of the TATA box (Plaschka et al , 2016). The observed differences could be a result of DNA bending by TBP in the Pol II PIC, which brings the DNA in close proximity to the C‐terminal cyclin domain (Murakami et al , 2013, 2015; Plaschka et al , 2015, 2016; He et al , 2016). Nevertheless, the overall conformation of the C‐terminal cyclin domain relative to the N‐terminal cyclin domain is conserved between TFIIB‐like factors (Fig 3C).…”