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. Author manuscript; available in PMC: 2016 Jul 22.
Published in final edited form as: FEBS Lett. 2015 Jul 2;589(16):2124–2130. doi: 10.1016/j.febslet.2015.06.028

Figure 6. Single-turnover kinetic analysis of DdiTLP3-catalyzed 5'-end repair of uniformly-labeled tRNAIleGAU(1).

Figure 6

DdiTLP3 (2 μM) was used to initiate reactions with uniformly-labeled C+4-start (left) or U+3-start (right) mt-tRNAIleGAU(1) in the presence of 0.1 mM ATP and 0.5 mM NTP (CTP or UTP or both, as indicated). Reactions were quenched at time points ranging from 10 s to 2 h by addition of EDTA and RNase T1, and then treated with phosphatase prior to resolution by 20% polyacrylamide, 8M urea PAGE. Bands corresponding to unreacted substrate (S) and 3'-5' addition products (arrows) are labeled according to the same colors shown in Figure 5 with the identity of the added nucleotide(s) shown in red. Smaller oligonucleotides (4-6-mers) produced from these reactions are not shown for clarity. The 11-mer (yellow labeled band) is well-resolved from unreacted substrate bands on this higher percentage gel and remains constant during the course of the reaction, as expected.