Single-molecule Study of Translation Initiation: Poly(A) RNA and Paip2 Act as Allosteric Regulators of Poly(A)-Binding Protein

DoctorWhen bound to the 3' multiple adenosine monophosphate [poly(A)] tail of mRNA, poly(A)-binding protein (PABP) modulates messenger RNA (mRNA) translation and stability through its association with various proteins. In this study, individual PABP molecules in real time were visualized by single-molecule Fluorescence Resonance Energy Transfer (smFRET). The single-molecule studies combined with biochemical experiments have revealed that PABP, containing four RNA recognition motifs (RRMs), adopts a conformation upon poly(A) binding in which RRM1 is in close proximity to RRM4. The RRM1-2-3-4 is unidirectionally aligned along 3' to 5' poly(A). This conformational change is due to the bending of the region between RRM2 and RRM3. PABP-interacting protein 2 (Paip2) actively disrupts the bent structure of PABP to the extended structure, resulting in the inhibition of PABP-poly(A) binding. These results suggest that the changes in the configuration of PABP induced by interactions with various effector molecules, such as poly(A) and Paip2, play pivotal roles in its function. In addition to a single PABP binding to poly(A), the study of the polymerization of PABP molecules on the poly(A) structure will provide to better understand the molecular mechanism of how PABP bound to poly(A) enhances the translation efficiency through direct interaction with eIF4G and stabilizes the mRNA structure from deadenylation of various exonucleases since 3’ poly(A) tail of mRNA can cover multiple PABP molecules. After post-transcriptional modification, most of the eukaryotic 3' poly(A) tail is modified with 250 nt long poly(A). This length of poly(A) tail covers about 10 PABP molecules, but the configuration of multiple PABP molecules bound to poly(A) is under investigation. In fact, the single-molecule study of nanopore force spectroscopy for the configuration of two PABP molecules on poly(A) proposed a cooperative binding of PABP molecules through the C-C domain interaction, but a liner conformation of RRMs on poly(A) that they assumed requires the opposite directionality of the two PABP molecules. However, the bent conformation of PABP can allow the direct interaction of C-terminus domains of two PABP molecules in the same directionality, which was shown by additional smFRET experiments and electrophoretic mobility shift assay