Analysis of GTP addition in the reverse (3′–5′) direction by human tRNA^His guanylyltransferase

5′-end RNA labeling with various GTP analogs by HsThg1. (A) Reaction scheme of single or multiple 5′-end RNA labeling by HsThg1 with 3′RF-A or 3′RF-C. HsThg1 adds single or multiple modified GTP analogs (XTP) to 5′-triphosphorylated 5′RF. (B) GTP analogs used in the 5′-end labeling reaction. Positions at which the GTP can be modified are indicated with Z (position 7) or M (2′OH). (C,D) Nucleotide addition by HsThg1 with various GTP analogs onto two-piece tRNA containing 3′RF-A (C) or 3′RF-C (D). The reaction mixture was incubated with HsThg1 and 1 mM modified GTP derivatives for 60 min and analyzed using urea-PAGE gels.