IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Folding mechanism of the intrinsically disordered dsRNA binding domain of Arabidopsis thaliana DCL1
Autor/es:
SUAREZ IP; BURDISSO P; HAILS G; RASIA RM
Lugar:
Zurich
Reunión:
Congreso; EUROMAR 2014; 2014
Resumen:
DCL1 is the ribonuclease that carries out miRNA biogenesis in plants. The enzyme has two tandem double stranded RNA binding domains (dsRBDs) in its C-terminus, which are essential for the enzyme function in vivo. By means of fluorescence anisotropy assays, we show that the first of these domains (DCL1-A) binds precursor RNA fragments when isolated, and cooperates with the second domain in the recognition of substrate RNA. Remarkably, despite showing RNA binding activity, DCL1-A is intrinsically disordered. We produced four different constructs of the protein, spanning the isolated domain and including surrounding regions. The domain is unstructured in every case. We explored different solution conditions and additives to test what could lead the domain to acquire an ordered structure, and found that it acquires a folded conformation when bound to its substrate dsRNA. We assigned ca. 90% of the backbone resonances corresponding to the free unfolded and bound folded protein. Analysis of NMR data of the free protein shows it transiently explores secondary structure elements on the C-term end that could be essential for its capability of binding to the substrate. We have calculated the structure of the folded protein in complex with dsRNA employing CS-Rosetta. The structure corresponds to a canonical dsRBD, bearing some differences. One of the three RNA binding regions is missing, but affinity for the substrate is not affected. Finally we found that in the presence of excess dsRNA some disordered protein remains in the mixture, but it is not the same as the free unfolded form. ZZ exchange experiments show that this unfolded form is in slow exchange with the folded form. Based on these results, we propose a binding mechanism in which the folding event appears to happen on the surface of the RNA, being preceded by the formation of a lower affinity complex with the unfolded species that is in fast exchange with the free protein