Reconstitution of CF IA from overexpressed subunits reveals stoichiometry and provides insights into molecular topology.
Yeast cleavage factor I (CF I) is an essential complex of five proteins that binds signal sequences at the 3' end of yeast mRNA. CF I is required for correct positioning of a larger protein complex, CPF, which contains the catalytic subunits executing mRNA cleavage and polyadenylation. CF I is composed of two parts, CF IA and Hrp1. The CF IA has only four subunits, Rna14, Rna15, Pcf11, and Clp1, but the structural organization has not been fully established. Using biochemical and biophysical methods, we demonstrate that CF IA can be reconstituted from bacterially expressed proteins and that it has 2:2:1:1 stoichiometry of its four proteins, respectively. We also describe mutations that disrupt the dimer interface of Rna14 while preserving the other subunit interactions. On the basis of our results and existing interaction data, we present a topological model for heterohexameric CF IA and its association with RNA and Hrp1.
Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111, United States.
James M B
JM
Gordon
Sergei
S
Shikov
Jason N
JN
Kuehner
Melissa
M
Liriano
Eunhee
E
Lee
Walter
W
Stafford
Mathias Bach
MB
Poulsen
Celia
C
Harrison
Claire
C
Moore
Andrew
A
Bohm
D015870:Gene Expression; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D039221:mRNA Cleavage and Polyadenylation Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
C569777:CF IA protein, S cerevisiae; C517046:Clp1 protein, S cerevisiae; C512636:HRP1 protein, S cerevisiae; C512637:PCF11 protein, S cerevisiae; D021122:Protein Subunits; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D039221:mRNA Cleavage and Polyadenylation Factors; C512638:RNA14 protein, S cerevisiae; C512635:RNA15 protein, S cerevisiae