Figure 4.

BtTLP catalyzes robust G₊₁ addition to 5′-truncated C₇₂-tRNA^His_ΔG+1. G₊₁ addition to 5′-³²P labeled-C₇₂tRNA^His_ΔG+1 was performed as described for G₋₁ addition assay above, using serial dilutions of yeast Thg1 (yThg1), BtTLP or MaTLP, as indicated, in the presence of 0.1 mM ATP and 1.0 mM GTP. The identity of the G₊₁p*GpC product was verified by migration with standards and RNase T2 digestion to release 3′-GMP (data not shown). The lower migrating products indicated by the bracket cannot be unequivocally identified due to the remote position of the labeled phosphate from the added nucleotide, but further digestions and comparison to known standards suggests that these are a mixture of further activation and addition products following G₊₁ addition.