FIG 6.

pRNA acceptor activity of GDP analogues in RNA capping with the VSV L protein. (A) The purified covalent L-pRNA intermediate (containing ³²P-labeled pAACAG) was incubated with GDP analogues for 1 min. The resulting capped RNAs released from the L protein were analyzed by 20% urea-PAGE followed by autoradiography. The autoradiogram is a representative of three independent experiments. Relative cap formation activities with GDP (defined as 100%) and GDP analogues are shown in the lower panel. Columns and error bars indicate the means and standard deviations, respectively. (B) Capped RNAs were eluted from gel pieces (excised from the region marked by an asterisk in panel A) and digested with nuclease P1 and CIAP. Cap structures were purified by DEAE Sephacel and analyzed together with standard GpppA and m⁷GpppA by PEI-cellulose TLC followed by autoradiography. Note that band intensities of purified cap structures did not match the radioactivities of the capped RNA products before purification shown in panel A.