ABSTRACT
New transcripts generated by RNA polymerase II (RNAPII) are generally processed in order to form mature mRNAs. Two key processing steps include a precise cleavage within the 3' end of the pre-mRNA, and the subsequent polymerization of adenosines to produce the poly(A) tail. In yeast, these two functions are performed by a large multi-subunit complex that includes the Cleavage Factor IA (CF IA). The four proteins Pcf11, Clp1, Rna14 and Rna15 constitute the yeast CF IA, and of these, Pcf11 is structurally the least characterized. Here, we provide evidence for the binding of two Zn2+ atoms to Pcf11, bound to separate zinc-binding domains located on each side of the Clp1 recognition region. Additional structural characterization of the second zinc-binding domain shows that it forms an unusual zinc finger fold. We further demonstrate that the two domains are not mandatory for CF IA assembly nor RNA polymerase II transcription termination, but are rather involved to different extents in the pre-mRNA 3'-end processing mechanism. Our data thus contribute to a more complete understanding of the architecture and function of Pcf11 and its role within the yeast CF IA complex.
Subject(s)
3' Untranslated Regions/genetics , RNA 3' End Processing/physiology , Saccharomyces cerevisiae Proteins/chemistry , Zinc/metabolism , mRNA Cleavage and Polyadenylation Factors/chemistry , Amino Acid Sequence , Binding Sites , Models, Molecular , Protein Binding , Protein Conformation , Protein Domains , RNA 3' End Processing/genetics , RNA Polymerase II/metabolism , RNA Precursors/metabolism , RNA, Fungal/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/physiology , Sequence Alignment , Sequence Homology, Amino Acid , Thermodynamics , mRNA Cleavage and Polyadenylation Factors/metabolism , mRNA Cleavage and Polyadenylation Factors/physiologyABSTRACT
Pcf11p and Clp1p form a heterodimer and are subunits of the Cleavage Factor IA (CF IA), a complex that is involved in the maturation of the 3'-end of mRNAs in Saccharomyces cerevisiae. The role of Clp1p protein in polyadenylation remains elusive, as does the need for ATP binding by Clp1p. In order to obtain structural details at atomic resolution of point mutants of Clp1p, we solved the crystal structure of Clp1-1p (G135R) point mutant complexed with Pcf11p (454-563) domain. The Clp1-1p-Pcf11p structure provides the atomic details for ATP loss while the point mutation preserves intact the Pcf11p interaction surface of Clp1p. This provides a rationale for the absence of phenotype in the yeast clp1-1 strain. Additionally, the structure allows for the description of an extended binding interface of Pcf11p with Clp1p which is likely to be S. cerevisiae specific.
