The Saccharomyces cerevisiae U2 snRNA:pseudouridine-synthase Pus7p is a novel multisite–multisubstrate RNA:Ψ-synthase also acting on tRNAs

Tests of the tRNA:Ψ1, Ψ13 and Ψ35-synthase activities in different yeast S10 extracts. In vitro transcribed RNA substrates labeled by incorporation of [α-³²P]ATP (yeast tRNA^Asp, A, yeast pre-tRNA^Tyr, B) or [α-³²P]UTP (yeast tRNA^Arg, C) were incubated with different S10 extracts in the conditions described in Materials and Methods. Extracts were prepared from cells of the WT BY4742 strain (WT), the isogenic ΔPUS7 strain, and the ΔPUS7 strain complemented with the WT or D256A variant PUS7 gene. The activity on yeast pre-tRNA^Tyr of an extract from the ΔPUS1 BY4742 strain was also tested. The tRNA^Asp and tRNA^Arg transcripts incubated in the same conditions but in the absence of S10 extract, were used as controls. After incubation, the transcripts were digested with T2 RNase and 3′NMPs were fractionated on TLC as described in Materials and Methods. The autoradiograms of the TLC plates are shown. Positions of the NMPs (Ap, Cp, Up, Gp) and ΨMP nucleotides were identified according to Keith (1995). Quantification of the Ψ residue formation was done by measuring the radioactivity in each spot with a PhosphoImager and the ImageQuant software.