Structure of a nucleotide-bound Clp1-Pcf11 polyadenylation factor

Abstract

Pcf11 and Clp1 are subunits of cleavage factor IA (CFIA), an essential polyadenylation factor in Saccahromyces cerevisiae. We have determined the structure of a ternary complex of Clp1 together with ATP and the Clp1-binding region of Pcf11. Clp1 contains three domains, a small N-terminal β sandwich domain, a C-terminal domain containing a novel α/β-fold and a central domain that binds ATP. The arrangement of the nucleotide binding site is similar to that observed in SIMIBI-class ATPase subunits found in other multisubunit macromolecular complexes. However, despite this similarity, nucleotide hydrolysis does not occur. The Pcf11 binding site is also located in the central domain where three highly conserved residues in Pcf11 mediate many of the protein–protein interactions. We propose that this conserved Clp1–Pcf11 interaction is responsible for maintaining a tight coupling between the Clp1 nucleotide binding subunit and the other components of the polyadenylation machinery. Moreover, we suggest that this complex represents a stabilized ATP bound form of Clp1 that requires the participation of other non-CFIA processing factors in order to initiate timely ATP hydrolysis during 3′ end processing.

Protein factors in pre-mRNA 3′-end processing

“…The structure of Clp1p confirms the similarity to other ATPases (Fig. 4C) [180]. The bound conformation of ATP suggests that Clp1p may not have ATPase activity, which is confirmed by biochemical assays in vitro.…”

“…(C). Crystal structure of Clp1p in complex with ATP and a peptide from Pcf11p [180]. A schematic model for the mammalian pre-mRNA 3′-end processing machinery.…”

The C terminus of Pcf11 forms a novel zinc-finger structure that plays an essential role in mRNA 3′-end processing

“…According to the BioGRID database, there are seven interacting partners for Pcf11 identified by at least two experimental approaches, although some of these interactions might be mediated by another factor: Clp1, Rna14, Rna15, Rpo21, Ysh1, Ydh1, and Yra1. The middle region of Pcf11 helps in the assembly and localization of Rna14/Rna15 and Clp1 to the 3 ′ -UTR (Noble et al 2007;Qu et al 2007;Gordon et al 2011). Yra1 putatively interacts with Pcf11 through a region N-terminal to the very C-terminal zinc finger to facilitate assembly of the pre-mRNA for recognition by the RNA export machinery (Johnson et al 2009(Johnson et al , 2011.…”

“…In yeast, these factors are organized into three complexes called cleavage factor IA (CF IA), cleavage factor II (CF II), and polyadenylation factor I (PF I). The structural basis for the activities of many individual proteins has been characterized (Noble et al 2005(Noble et al , 2007Paulson and Tong 2012;Xiang et al 2014), but how these activities are integrated into the complete processing apparatus and how different steps are orchestrated and coordinated remain unclear at the molecular level.…”

“…The N-terminal and Clp1 interaction regions have been characterized both structurally and functionally. Namely, mutations disrupting the Pcf11/Clp1 interaction cause defects in growth and 3 ′ -end processing (Noble et al 2007;Ghazy et al 2012). This region might also maintain interactions with Yra1, an mRNA export factor (Johnson et al 2009(Johnson et al , 2011.…”