Cryo-EM structure of a mammalian RNA polymerase II elongation complex inhibited by α-amanitin

Abstract

RNA polymerase II (Pol II) is the central enzyme that transcribes eukaryotic protein-coding genes to produce mRNA. The mushroom toxin α-amanitin binds Pol II and inhibits transcription at the step of RNA chain elongation. Pol II from yeast binds α-amanitin with micromolar affinity, whereas metazoan Pol II enzymes exhibit nanomolar affinities. Here, we present the high-resolution cryo-EM structure of α-amanitin bound to and inhibited by its natural target, the mammalian Pol II elongation complex. The structure revealed that the toxin is located in a pocket previously identified in yeast Pol II but forms additional contacts with metazoan-specific residues, which explains why its affinity to mammalian Pol II is ∼3000 times higher than for yeast Pol II. Our work provides the structural basis for the inhibition of mammalian Pol II by the natural toxin α-amanitin and highlights that cryo-EM is well suited to studying interactions of a small molecule with its macromolecular target.

Keywords: RNA polymerase; RNA polymerase II; cryo-electron microscopy; structural biology; transcription.

Figure 1.

Pig (S. scrofa) Pol II purification, in vitro RNA extension assay, and exemplary 2D classes of the data set. A, SDS-PAGE analysis of the Pol II–hGdown1 complex. B, the reconstituted Pol II–hGdown EC is active in RNA extension and inhibited by α-amanitin. In the absence of α-amanitin (upper panel), two uridine residues were incorporated into the RNA of the scaffold upon incubation with 100 mm UTP, as expected from the presence of two templating adenine bases downstream. In the presence of α-amanitin (lower panel), nucleotide addition is slowed down, and addition of only one uridine residue was observed, as expected from impaired Pol II translocation. C, representative 2D classes generated from the cryo-EM data set.

Figure 2.

Cryo-EM structure of mammalian Pol II EC bound by α-amanitin. A, nucleic acid scaffold is depicted schematically. Filled and unfilled circles represent modeled and not modeled nucleotides, respectively. The nucleotide-binding site (red dashed circle), bridge helix (green), the catalytic metal ion A (pink), trigger loop (brown), and α-amanitin (orange) are indicated. The color code is used throughout. B, overview of the structure. Pol II is shown as a silver ribbon model, and other elements are colored as in A. C, electron density for α-amanitin (orange mesh) in three different views. Important contact moieties with Pol II are indicated. Nitrogen, oxygen, and sulfur atoms are blue, red, and yellow, respectively.

Figure 3.

Interactions of mammalian Pol II with α-amanitin. A, sequence alignment of residues forming the α-amanitin-binding pocket in RPB1 between various metazoan species and the yeast S. cerevisiae (bottom row). The red boxes indicate amino acid residues that form metazoan-specific interactions with α-amanitin. Helices α21 to α24, bridge helix, and trigger loop are indicated at the bottom of the sequence alignment. B, schematic overview of Pol II–amanitin interactions. The chemical structure of α-amanitin is shown in orange. RPB1 residues conserved over eukaryotes are labeled in black, whereas metazoan-specific amanitin-interacting residues are labeled in red. The green dashed lines indicate hydrogen bonds, whereas black dashed lines show other interactions. C, surface representation of the amanitin-binding Pol II pocket. Positively and negatively charged surfaces are in blue and red, respectively. The bridge helix, trigger loop, and RPB1 residue Ser⁷⁸² are indicated.

Figure 4.

Extra hydrogen bonds in mammalian and binding pocket mutation analysis. A, two metazoan-specific hydrogen bonds are indicated with green dashed lines, and the corresponding bond lengths are indicated between α-amanitin and mammalian RPB1. B, modeling of site-specific mutations in the α-amanitin–binding pocket that confer resistance to α-amanitin in Mus musculus. The Pol II model is shown with gray sticks, whereas the mutated amino acids are shown with magenta sticks.