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. 2013 Aug 22;41(21):9825–9838. doi: 10.1093/nar/gkt741

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© The Author(s) 2013. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — Post-transfer editing and mis-aminoacylation of CatRNA^Leu by CaLeuRS and CaLeuRS-D422A. (A) A representative graph showing hydrolysis of Nva-[³²P]CatRNA^Leu by CaLeuRS and CaLeuRS-D422A. Nuclease S1-generated Nva-[³²P]AMP (reflecting Nva-[³²P]CatRNA^Leu) and [³²P]AMP (reflecting free [³²P]CatRNA^Leu) were separated by TLC. A control reaction represented the spontaneous hydrolysis of Nva-[³²P]CatRNA^Leu without the addition of enzyme. (B) Analysis of post-transfer editing of Nva-[³²P]CatRNA^Leu in (A). (C) A representative graph showing mis-charging of [³²P]CatRNA^Leu with non-cognate Nva (left) or ABA (right). Free [³²P]CatRNA^Leu and mis-charged [³²P]CatRNA^Leu are represented by [³²P]AMP and Nva-[³²P]AMP or ABA-[³²P]AMP, respectively. Known amounts of [α-³²P]ATP were serially diluted and loaded onto the TLC plate after separation for quantification. (D) Quantitative analysis of Nva-[³²P]CatRNA^Leu or ABA-[³²P]CatRNA^Leu generated by CaLeuRS (black square) and CaLeuRS-D422A (black up-pointing triangle) or ABA-[³²P]CatRNA^Leu by CaLeuRS (white up-pointing triangle) and CaLeuRS-D422A (white down-pointing triangle) in (C).