Figure 2.

BVP leaves a 5′-monophosphate end. The RNA triphosphatase assay was performed with [α-³²P]ATP-terminated trinucleotide RNA (label is indicated by an asterisk). After incubation for 10 min at 30°C, the reaction mixture was analyzed by (A) PEI–cellulose TLC or (B) 25% polyacrylamide gel electrophoresis. The positions of ADP- and AMP-terminated RNA trinucleotides were determined by using ADP- and AMP-terminated trimers as standards (not shown). In A, RNA substrate was incubated with the following: buffer (lane 1), the indicated amount of BVP (BVP, lanes 2–4), an active site mutant of BVP [BVP(C119S), lanes 5–7], 1 unit of CIP (lane 8), the C. elegans capping enzyme triphosphatase (Cel-1[1–236], lanes 9–11), or the corresponding active site mutant [Cel-1[1–236](C124S), lanes 12–14]. Reactions in lanes 1–5 of B were identical to those in lanes 1–4 and 8 of A.