Legends of supplementary figures
Figure S1. The elution profiles of PAZ domain alone and PAZ-9-mer RNA
complex on superdex 75. Blue and red curves strand for the absorbance at 280
and 260 nm, respectively. The protein alone has a higher absorbance at 280 nm
than that at 260 nm, whereas the protein-RNA complex shows reverse behavior.
Compared to the protein standards, the elution peak of PAZ alone corresponds
to that of an 18 kD species, suggestive of a monomeric protein. By contrast, the
elution peak of the PAZ-9-mer RNA complex corresponds to a 49 kD species,
suggestive of 2:2 complex formation.
Figure S2. Stereo view of structural superposition of PAZ domains. Red-colored
backbone represents our crystal structure of the PAZ domain from human eIF2cI
bound by siRNA-like duplex. Green-colored backbone represents the structure of
free PAZ domain of Drosophila Argonaute I solved by NMR (Yan et al, 2003),
which aligns against our structure with1.44 Å root-mean-square deviation (rmsd)
for 109 aligned Ca atoms. Blue-colored backbone represents the structure of free
PAZ domain of Drosophila Argonaute II solved by NMR (Lingel et al, 2003),
which aligns against our structure with 1.79 Å rmsd for 87 aligned Ca atoms.
Superposition was made in PyMOL using a combination of sequence and
structural alignment. The C-terminal end of our PAZ construct is extended from
the core scaffold and does not form any secondary structure element. The
interaction of the C-terminus of our PAZ construct with RNA may nevertheless
have functional implications in the context of the full-length protein, given that it
could form a short -sheet with a longer N-terminal segment, as observed in the
PAZ domain of Drosophila Argonaute I (Yan et al, 2003).
Figure S3. The sequence alignment of PAZ domains. The aligned sequences
(genebank id) are in the order of Human eIF2C1 (6912352), Human eIF2C2
(29171734), Drosophila Argonaute 1 (17647145), Drosophila Argonaute 2
(24664668),
Human
HIWI
(33563234),
Drosophila
PIWI
(17136736),
Tetrahymena TWI1(21671127), Human Dicer (29294651), Drosophila Dicer1
(17738129), and Arabidopsis Dicer (15223286). The secondary structure
diagram is shown on the bottom. Dots denote disordered regions. Conserved
residues are shaded in blue and invariant residues in red. Triangles denote
residues directly interacting with RNA.