Figure 2.

Analysis of the activity of purified GST YGR024c fusion protein. (A) tRNA substrate specificity of YGR024p. Here, 0.4 μM p-tRNA^Phe (lanes a-c) or 0.4 μM p-tRNA^His (lanes d-f) was incubated in reaction mixtures containing 100 μM ATP, 0.6 μM [α-³²P]GTP, and GST dialysis buffer (lanes a,d), 0.5 μM GST-YGR024p (lanes b,e), or 2.4 μg/μL S100 extract (lanes c,f), and tRNA was resolved by PAGE as described in Materials and Methods. Lane g, [5′-³²P] tRNA^His. (B) Phosphatase treatment of guanylylated tRNA^His. Gel-purified tRNA product from reaction with GST-YGR024p was incubated with calf intestinal phosphatase (CIP), and products were resolved by thin layer chromatography as described in Materials and Methods. a, tRNA incubated on ice; b, tRNA on ice in CIP buffer; c, tRNA in CIP buffer at 37°C; d, tRNA incubated with CIP in CIP buffer at 37°C; e, ³²P-labeled inorganic phosphate. (C) Partial RNase T1 digestion of guanylylated tRNA^His. Gel-purified guanylylated tRNA^His and labeled tRNA standards were incubated with RNase T1 under partial digestion conditions, as described in Materials and Methods, and products were resolved by 12% PAGE. a, [5′-³²P]tRNA^His 76-mer, beginning with G_-1 (p^*76mer); b, [5′-³²P]tRNA^His 75-mer, beginning with G₊₁ (p^*75mer); c,d, p-tRNA^His substrate guanylylated with GST-YGR024p and GST-YDL076p, respectively, in the presence of 100 μM ATP and [α-³²P]GTP; e, 75-mer tRNA^His 3′-labeled with ^*pCp. Numbers on the left and right indicate the length of partially digested fragments in lanes a and e, respectively.