Near-atomic resolution visualization of human transcription promoter opening

Abstract

In eukaryotic transcription initiation, a large multi-subunit pre-initiation complex (PIC) that assembles at the core promoter is required for the opening of the duplex DNA and identification of the start site for transcription by RNA polymerase II. Here we use cryo-electron microscropy (cryo-EM) to determine near-atomic resolution structures of the human PIC in a closed state (engaged with duplex DNA), an open state (engaged with a transcription bubble), and an initially transcribing complex (containing six base pairs of DNA–RNA hybrid). Our studies provide structures for previously uncharacterized components of the PIC, such as TFIIE and TFIIH, and segments of TFIIA, TFIIB and TFIIF. Comparison of the different structures reveals the sequential conformational changes that accompany the transition from each state to the next throughout the transcription initiation process. This analysis illustrates the key role of TFIIB in transcription bubble stabilization and provides strong structural support for a translocase activity of XPB.

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).…”

“…In sequential steps, the PIC transitions from a closed complex (CC), in which the DNA is still double‐stranded, to an open complex (OC), in which the double‐stranded DNA in the vicinity of the transcription start site is melted. Finally, an initially transcribing complex (ITC) forms when short RNA is synthesized just before RNA polymerase enters the elongation phase (Plaschka et al , 2015; Sainsbury et al , 2015; He et al , 2016). …”

Profile of Eva Nogales

“…"Now we have to start adding in the regulation," she says, by incorporating a multitude of molecules that affect transcription. She notes that her laboratory has already visualized a preinitiation complex with more than 30 proteins in several functional states (9).…”

What have single-molecule studies taught us about gene expression?

“…It has also been shown that scrunching in RNAP helps to determine the exact DNA base from which to begin transcription (Robb et al 2013;Winkelman et al 2016). Recent cryo-EM structures of human PICs show breaks in electron density on the nontemplate DNA strand in the RP itc , and this was proposed to be disordered due to possible scrunching within RP itc (He et al 2016). Thus, it appears that scrunching could be a universal scanning mechanism in both prokaryotes and eukaryotes that helps position the correct starting base in the catalytic center of polymerase.…”