title: Nanopore detachment kinetics of poly(A) binding proteins from RNA molecules reveals the critical role of C-terminus interactions.
abstract: The ubiquitous and abundant cytoplasmic poly(A) binding protein (PABP) is a highly conserved multifunctional protein, many copies of which bind to the poly(A) tail of eukaryotic mRNAs to promote translation initiation. The N-terminus of PABP is responsible for the high binding specificity and affinity to poly(A), whereas the C-terminus is known to stimulate PABP multimerization on poly(A). Here, we use single-molecule nanopore force spectroscopy to directly measure interactions between poly(A) and PABPs. Both electrical and biochemical results show that the C-C domain interaction between two consecutive PABPs promotes cooperative binding. Up to now, investigators have not been able to probe the detailed polarity configuration (i.e., the internal arrangement of two PABPs on a poly(A) streak in which the C-termini face toward or away from each other). Our nanopore force spectroscopy system is able to distinguish the cooperative binding conformation from the noncooperative one. The ∼50% cooperative binding conformation of wild-type PABPs indicates that the C-C domain interaction doubles the cooperative binding probability. Moreover, the longer dissociation time of a cooperatively bound poly(A)/PABP complex as compared with a noncooperatively bound one indicates that the cooperative mode is the most stable conformation for PABPs binding onto the poly(A). However, ∼50% of the poly(A)/PABP complexes exhibit a noncooperative binding conformation, which is in line with previous studies showing that the PABP C-terminal domain also interacts with additional protein cofactors.
authors.affiliation: Department of Biomedical Engineering, Boston University, Boston, Massachusetts, USA.
authors.forename: Jianxun
authors.initials: J
authors.lastname: Lin
authors.forename: Marc
authors.initials: M
authors.lastname: Fabian
authors.forename: Nahum
authors.initials: N
authors.lastname: Sonenberg
authors.forename: Amit
authors.initials: A
authors.lastname: Meller
mesh_terms: D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D058608:Nanopores; D039102:Poly(A)-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D012313:RNA / Q000378:metabolism*; D013997:Time Factors
chemical_list: D039102:Poly(A)-Binding Proteins; D012313:RNA