Figure 6. Effects of TOM1 and ARL8 coexpression on RNA 5′ capping functions of ToMV 130K protein.

(A) A possible mechanism of RNA 5′ capping by tobamovirus replication proteins. The phosphate group at the α position of GTP is shown in red. The 5′ phosphate groups of the substrate RNA are shown in blue. See text for details. AdoHcy: S-Adenosyl-L-homocysteine. (B) Effects of the coexpression of TOM1 and ARL8 on RNA 5′ capping and guanylation of ToMV 130K protein expressed in yeast. P15 membrane fractions were prepared from the indicated yeast and BY-2 cells. The fractions were treated with LPC and subjected to immunopurification with anti-FLAG antibody [27]. The protein samples were prepared so that the concentration of the replication proteins was similar (samples for lanes 1 and 6 were prepared and analyzed in the same way as for those for lanes 2 and 7, respectively). The samples were incubated at 25°C for 60 min with [α-³²P]GTP, AdoMet and uncapped ToMV (1–30) RNA with 5′-triphosphate or 5′-diphosphate. After the reaction, RNA was purified by phenol extraction and separated by 8 M urea-9% PAGE. Fractions of the purified RNA samples were treated with TAP and analyzed similarly. ³²P-labeled RNA was visualized with an image analyzer (BAS2500, Fujifilm). The protein samples were also subjected to immunoblot analysis with anti-ToMV replication protein antibodies and protein guanylation assay as described in the Materials and Methods.