Figure 5.

In vitro 2′-O-methylation activity of the reconstituted RNP complex (RNP3). (A) Kinetics of labeled methyl incorporation into the H.volcanii (circles) or P.abyssi (squares) pre-tRNA^Trp transcripts. (B) Characterization of the 2′-O-methylation sites by 2D-TLC of an RNase T2 digest of in vitro synthesized H.volcanii pre-tRNA^Trp. The transcript was labeled by incorporation of [α-³²P]UTP and [α-³²P]CTP (top and bottom, respectively) in the absence and presence (left and right panels, respectively) of the three recombinant proteins supplemented with SAM. Chromatographic separation was performed with system B. (C) As in (B) using in vitro synthesized P.abyssi pre-tRNA^Trp. The transcript was labeled by incorporation of [α-³²P]CTP and [α-³²P]ATP (top and bottom, respectively) in the absence and presence (left and right panels, respectively) of the three recombinant proteins supplemented with SAM.