Figure 6.

The central LCR of Fip1₂₂₆ is important for CPF function. (A) SDS-PAGE of purified CPF alongside CPF(Fip1Δ110–180). Deletion of the central LCR in Fip1 does not affect the assembly and purification of CPF. The composition and stoichiometries of CPF subunits are similar in both samples. (B) Denaturing gel electrophoresis of coupled cleavage and polyadenylation assays of a CYC1 3′ UTR containing a 5′-FAM and a 3′-A647 label. Each reaction contained 50 nM CPF or CPF(Fip1Δ110–180), 100 nM CYC1 3′-UTR, and 300 nM CF IA and IB. The cleavage activity of CPF is compromised in the absence of the central LCR in Fip1. This gel is representative of experiments performed twice. (C) Polyadenylation activity of wild-type CPF compared with CPF(Fip1Δ110–180). The RNA contains a 5′-FAM label. This gel is representative of experiments performed twice. (D) Model for CPF. The central IDR of Fip1 flexibly tethers Pap1 to the complex. Additionally, IDRs within other subunits in each of the modules may allow flexibility to permit conformational remodeling.