Figure 5.

BtTLP adds all four possible templated N₊₁ nucleotides to 5′-truncated tRNA^His variants (N₇₂-tRNA^His_ΔN+1). (A) N₊₁ addition assays were performed using the same assay described in Figure 2, but with 5′-³²P labeled tRNA^His variants missing a +1 nt and containing each of the four possible N₇₂ nucleotides to serve as the template for +1 nt addition (see tRNA diagram). The single N₊₁ addition products produced by the relevant nuclease treatment are indicated by arrows. The full-length tRNAs generated following N₊₁ addition are each substrates for further activation and/or N₋₁ addition reactions; products of these reactions are indicated by asterisks to the right of the image, but these products are not further identified since the remote position of the labeled phosphate (between N₊₁ and G₊₂ nucleotides) does not readily permit identification by RNase T2 digestion. (B) RNase T2 digestion of reactions from (A) confirms each of the four N₊₁ nucleotides added to 5′-truncated tRNA^His_ΔN+1 substrates. RNase T2 products were resolved by PEI-cellulose TLC in 0.5 M formate, pH 3.5; positions of each 3′-³²P labeled mononucleotide products (Cp*, Up*, Ap* and Gp*) were identified based on the migration of cold NMP standards. 5′-activated N₊₁ addition products generated from G₊₁ and A₊₁ addition reactions are indicated by NppGp* and NppAp*, respectively.