Structure and function of the initially transcribing RNA polymerase II–TFIIB complex

Abstract

The general transcription factor (TF) IIB is required for RNA polymerase (Pol) II initiation and extends with its B-reader element into the Pol II active centre cleft. Low-resolution structures of the Pol II-TFIIB complex 1,2 indicated how TFIIB functions in DNA recruitment, but they lacked nucleic acids and half of the B-reader, leaving other TFIIB functions 3,4 enigmatic. Here we report crystal structures of the Pol II-TFIIB complex from the yeast Saccharomyces cerevisiae at 3.4 Å resolution and of an initially transcribing complex that additionally contains the DNA template and a 6-nucleotide RNA product. The structures reveal the entire B-reader and proteinnucleic acid interactions, and together with functional data lead to a more complete understanding of transcription initiation. TFIIB partially closes the polymerase cleft to position DNA and assist in its opening. The B-reader does not reach the active site but binds the DNA template strand upstream to assist in the recognition of the initiator sequence and in positioning the transcription start site. TFIIB rearranges active-site residues, induces binding of the catalytic metal ion B, and stimulates initial RNA synthesis allosterically. TFIIB then prevents the emerging DNA-RNA hybrid duplex from tilting, which would impair RNA synthesis. When the RNA grows beyond 6 nucleotides, it is separated from DNA and is directed to its exit tunnel by the B-reader loop. Once the RNA grows to 12-13 nucleotides, it clashes with TFIIB, triggering TFIIB displacement and elongation complex formation. Similar mechanisms may underlie all cellular transcription because all eukaryotic and archaeal RNA polymerases use TFIIB-like factors 5 , and the bacterial initiation factor sigma has TFIIB-like topology 1,2 and contains the loop region 3.2 that resembles the B-reader loop in location, charge and function [6][7][8] . TFIIB and its counterparts may thus account for the two fundamental properties that distinguish RNA from DNA polymerases: primer-independent chain initiation and product separation from the template.Our previous X-ray analysis of the Pol II-TFIIB complex at 4.3 Å resolution provided a partial backbone model of TFIIB 1 . To obtain a complete and atomic structure, we co-crystallized Pol II with a TFIIB variant lacking the mobile amino-terminal tail and carboxy-terminal cyclin fold (Methods, Supplementary Table and Supplementary Fig. 1). The resulting Pol II-TFIIB structure at 3.4 Å resolution provides details of the interactions of the four TFIIB domains with Pol II: the B-ribbon with the dock, the B-core N-terminal cyclin fold with the wall, the B-reader helix with the RNA exit tunnel, and the B-linker helix with the coiled-coil of the clamp (Fig. 1).The structure reveals the entire course of the TFIIB polypeptide chain through the Pol II cleft, including the previously lacking 1 B-reader loop (residues 67-79) and a new 'B-reader strand' (residues 80-83). The B-reader loop does not reach the active site, but instead interacts with the Pol II rudder and fork loop ...

RNA polymerase I structure and transcription regulation

“…Factors may bind in the pore, as observed for TFIIS 14 and Gfh1 (ref. 25), or near the RNA exit channel, as observed for Rrn3 (refs 29, 34), the Pol II initiation factor TFIIB that stimulates RNA chain initiation allosterically 47 , and the Pol II coactivator Mediator 48 . The bacterial regulator catabolite activator protein 49 and the growth regulator ppGpp also bind at the RNA exit channel, and the latter was suggested to influence polymerase activity by modulating the coreshelf interface 50 .…”

“…c, De novo RNA synthesis activity on tailed DNA template. Assays were performed as described 47 using the DNA template shown. After running a 20% acrylamide UREA gel, de novo synthesized, radioactive RNA was detected by phosphoimaging.…”

Structural insights into transcription initiation by yeast RNA polymerase I

“…However, the position of the Rrn7 cyclin domains relative to Pol I is very different. In the Pol II‐TFIIB crystal structures (Kostrewa et al , 2009; Liu et al , 2010; Sainsbury et al , 2013) and in the Pol II PIC (Plaschka et al , 2016), the N‐terminal cyclin domain is located in close proximity to the Pol II wall and the C‐terminal cyclin domain of TFIIB is positioned next to the Rpb2 protrusion and Rpb12. In contrast, in the Pol I PIC, both Rrn7 cyclin domains are located farther away from Pol I and do not directly contact Pol I.…”

“…TFIIB‐like factors including TFIIB and Brf2 (a TFIIB‐like factor present only in vertebrates) bind DNA utilizing the interfaces formed by helix α7 and a turn between helices α4 and α5 (Sainsbury et al , 2013; Gouge et al , 2015). Despite its different position, the N‐terminal cyclin domain of Rrn7 also uses this interface (Fig 3C).…”

Conserved architecture of the core RNA polymerase II initiation complex

“…We have previously modelled the architecture of the core Pol II initiation complex 9 by structural superposition of our Pol II-TFIIB crystal structures 10,11 with a Pol II-TFIIF complex model obtained by XL-MS 12 . However, the model awaited experimental confirmation because both TFIIF and TFIIB are modular factors with flexible domains that may be repositioned on complex assembly.…”