Fig. 3

Putative catalytic site and cleavage model of SLFN13-N. a Structural comparison between the C-lobe of rSLFN13_14–353 and the DNase I subdomain of RNase E (PDB ID 2BX2). For RNase E, the portion that is structurally aligned to rSLFN13_14–353 C-lobe is shown in blue, and the rest part in light blue. b The electrostatic surface potentials of rSLFN13_14–353, coloured from red (negative) to blue (positive). Two negatively charged patches and the conserved positively charged area are indicated. Positions of the residues tested in c are specified. c tRNA cleavage assay for rSLFN13_1–353 mutants of conserved charged residues in the presence of Mg²⁺. For each sample, 500 nM protein was used. d Comparison between the putative nucleolytic active site of rSLFN13-N and the actives sites of RNase E (2BX2) and RNase III (2EZ6). Note the similar tripod architecture of the three residues for each protein, which are shown as ball-and-stick models. e Cleavage assay of hSLFN13_1–355 mutants at the putative active site. E208A, E213A and D251A of hSLFN13-N correspond to E205, E210 and D248 of rSLFN13-N, respectively. f Schematic drawing of SLFN13 cleaving a tRNA. The U-pillow-shaped SLFN13 N′-domain embraces the acceptor stem of the tRNA. The 3′ tail that is to be cleaved off the tRNA by SLFN13-N is coloured purple. g Structural model of SLFN13-N manipulating tRNA. rSLFN13_14–353 is shown as surface representations and the subdomains are colour-specified. The putative active site is coloured red. The coordinate of tRNA^Gly (5E6M, excerpted) is used and coloured orange. SLFN13-N clamps the acceptor stem of tRNA and may have no contact with other parts of the tRNA. h Close view of SLFN13-N accommodating the acceptor stem. The surface positively charged residues of rSLFN13_14–353 tested in c are coloured blue. Part of the tRNA is removed for clarity. The 5′ and 3′ ends of the tRNA are indicated. The phosphate atom of the 66th nucleotide of tRNA is shown as a sphere