Structural basis of transcription: -Amanitin–RNA polymerase II cocrystal at 2.8 Å resolution

T structure of 10-subunit 0.5-MDa yeast RNA polymerase II (pol II), recently determined at 2.8 Å resolution, reveals the architecture and key functional elements of the enzyme (1). The two largest subunits, Rpb1 and Rpb2, lie at the center, on either side of a nucleic acid-binding cleft, with the many smaller subunits arrayed around the outside. Rpb1 and Rpb2 interact extensively in the region of the active site and also through a domain of Rpb1 that lies on the Rpb2 side of the cleft, connected to the body of Rpb1 by an -helix that bridges across the cleft. Proof that nucleic acids bind in the channel comes from the molecular replacement solution of a transcribing pol II complex at 3.3 Å resolution (2). This structure shows the template DNA unwinding some three residues before the active site, followed by nine base pairs of DNA–RNA hybrid. Adjacent regions of Rpb1 and Rpb2 form a highly complementary surface, resulting in extensive DNA–RNA hybrid–protein interaction. The ‘‘bridge’’ helix seems to play an important role, binding to both the second and third unpaired DNA bases and also to the coding base, paired with the first residue of the RNA. Comparison of the pol II structure in different crystal forms shows a division of the enzyme in several mobile elements that my facilitate DNA and RNA movement during transcription. Comparison of the pol II structure with that of the related bacterial RNA polymerase (3) suggests mobility of the bridge helix as well (2). The pol II structures open the way to many lines of investigation. Structures of cocrystals of pol II with interacting molecules can be solved, the full power of site-directed mutagenesis can be brought to bear on the transcription mechanism, and so forth. Here we report the structure of a cocrystal of pol II with the most potent and specific known inhibitor of the enzyme, -amanitin. The active principle of the ‘‘death cap’’ mushroom, -amanitin blocks both transcription initiation and elongation (4–6). The structure of the cocrystal suggests that -amanitin interferes with a protein conformational change underlying the transcription mechanism.