Nucleotide addition reaction by HsThg1 onto a two-piece tRNAHis. (A) Reaction scheme of a nucleotide addition reaction with natural NTPs and various GTP analogs (XTP) and a 5′-triphosphorylated RNA fragment (5′RF) with a 3′-side RNA fragment (3′RF) containing adenine or cytosine opposite the −1 position (Y = A or C). (B,C) Primer/template assay for simultaneously measuring the kinetics of nucleotide addition reactions by HsThg1 with two types of two-piece tRNA (Y = A or C). The reactions shown are time courses of activity with 10 µM HsThg1 and 0.1 mM GTP in excess over two-piece tRNA; aliquots from each time point were analyzed on urea-PAGE gels. RNA fragments were detected by SybrGold staining. Asterisk indicates upper bands which likely correspond to the G−2 addition to 5′RF/3′RF-A. (D) Time course experiments of the nucleotide addition reaction of HsThg1 with 3′RF-A (blue) or 3′RF-C (red). The bars in the graphs are SD of more than two independent experiments.
