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. 2019 Mar 27;47(9):4814–4830. doi: 10.1093/nar/gkz190

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© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 4. — Analysis of tRNA^Sec interaction with CtKti12_NTD. (A) (Top left): Structural model of tRNA-bound CtKti12_NTD based on superimposition with MjPSTK_NTD (3ADB), r.m.s.d. = 3.8 Å². tRNA^Sec is shown in grey and MjPSTK_CTD in yellow. The 3′ end of the superimposed tRNA^Sec reaches to the active site of CtKti12_NTD. Surface charge distribution of CtKti12_NTD is shown in blue and red. Positively charged regions (blue) coincide with a modelled contact surface with tRNA^Sec. Color coding and scale are identical to Figure S5. (Bottom right): The close up of the model shows the proposed tRNA interaction surface at the NTD, which is shown in light blue, acceptor stem of tRNA^Sec is gray. Mutants with significantly downregulated ATPase activity are shown in red, mutants with moderate ATPase properties in orange. Hydrolytic activity after mutating residues shown in green does not differ significantly from WT protein. The gatekeeping R213 residue (asterisk) is shown in violet. (B) Analyses of hydrolytic properties of tRNA interaction surface mutants using the malachite green assay. Calculated standard deviations are shown (n = 3). Color coding as in (A). (C) tRNA-independent ATP hydrolysis of mutants within tRNA-interaction interface on the NTD using the malachite green assay. The results were normalized to the tRNA^Sec-induced hydrolysis of WT protein and calculated standard deviations are shown (n = 3). (D) Binding affinity of tRNA-interface mutants to the bulk yeast tRNA. Samples were resolved on a 5% native PAGE. As a loading control, samples were also analyzed by denaturing SDS-PAGE stained with Coomassie. tRNA was stained using SYBR-Gold.