Disordered Motifs and Domains in Cell Control
Poster Session I
16-POS
Board 16
Intrinsically Disordered dsRNA Binding Domain of
Arabidopsis thaliana
DCL1 Folds in the
Presence of Substrate RNA
Irina P. Suarez
1
, Guillermo Hails
1
, Diego F. Gauto
1
, Matthieu P.M.H. Benoit
2
, Jèrôme
Boisbouvier
2
, Rodolfo M. Rasia
1
.
1
Institute for Molecular and Cellular Biology of Rosario, Rosario, Santa Fe, Argentina.
2
Institute
for Structural Biology, Grenoble, France.
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 to test what could lead the domain to acquire an ordered structure, and found that it
folds when bound to its substrate dsRNA. By acquiring a set of standard NMR spectra, 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 in the presence of excess dsRNA we observe an intermediate unfolded bound
species. 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 and discuss functional
implications.
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