Figure 4. Enzyme titration assay for 5'-end repair with 5'-³²P-labeled tRNA^Ile_GAU(1).

The C₊₄-start (A) or U₊₃-start (B) substrates were incubated with five-fold serial dilutions (2.7 μM to 0.2 nM) of each of the four TLP enzymes, as indicated, for two hours. Reactions were quenched by addition of EDTA and RNase T1 and the resulting oligonucleotides were resolved by 12% polyacrylamide, 7 M urea PAGE. The tRNA diagrams show the complete tRNA sequence, with the expected labeled oligonucleotide derived from unreacted substrate indicated by the green line; the band corresponding to this oligonucleotide is marked by the red (S) on the gels (as seen in lane -, which is the no enzyme control lane for each substrate). Reaction products corresponding to 3'-5' addition of the indicated NTPs are indicated by arrows, with expected sequences of the added nucleotides shown in red for each band. The red arrows indicate fully-repaired 5'-ends, and the dashed arrow indicates nucleotides added beyond the +1 (full-length) position. Lanes corresponding to reactions performed at 22 nM of the indicated TLP are marked with red stars for comparison between the two substrates.