Figure 2.
Mpe1 contacts the polyadenylation signal (PAS) in RNA and stimulates polyadenylation
(A) Sequence of the CYC1 RNA substrate. The full sequence is “uncleaved” CYC1.
(B) Cryo-EM map (transparent surface) and model (sticks) of PAS RNA in the polymerase module-Mpe1-RNA map.
(C) Contacts between the PAS of CYC1 RNA (gray) and Yth1 (pink) and Mpe1 (orange). P215 of Mpe1 contacts A2 via a CH-π interaction. Blue dashed lines show hydrogen bonds.
(D) The U−1 nucleotide (sticks and transparent surface) sits in an open pocket on Yth1 (magenta surface) and makes hydrogen bonds to the main chain of I65 and E82.
(E) Polyadenylation activity of polymerase module without or with Mpe1. Left, SDS-PAGE of purified complexes. Right, polyadenylation reactions using a 5′ FAM-labeled precleaved CYC1 RNA substrate (shown schematically with a black rectangle), analyzed by urea-PAGE. CF IA and CF IB were not included in these reactions.