Figure 6. Methylation status of the stalled transcripts.

(A) In vitro transcription reactions were reconstituted with 5 µg of N-RNA template, 2 µg of purified P, and 4 µg of the recombinant L protein in the absence of UTP, but in the presence of ³H-labeled S-adenosyl-L-methionine. Purified RNA was analyzed on a 20% polyacrylamide gel followed by autoradiography. The identity of the template used for the IVT reactions are shown at the top of the panel. Where indicated, the products of transcription were digested with tobacco acid pyrophosphatase (TAP) which cleaves the cap-structure. The transcripts between 31 and 40-nt are indicated. (B) The autoradiograms from two independent experiments equivalent to that shown in panel A, were analyzed by densitometric scanning and the band intensities measured. A graph showing the % of the total methylated RNA that remains following TAP cleavage is shown. (C) Total RNA from stalled in vitro transcription reactions performed in the presence of ³H-SAM was quantified by scintillation counting before and after TAP cleavage. A graph showing the % of total radioactivity measured by scintillation counting following TAP cleavage of the purified RNA is shown.