and analyzed for its Y content as described previously (20). In brief, each RNA sample was hydrolyzed by nuclease P1 or RNase T2 and the resulting nucleotides were analyzed by 1D-(20 cm) or 2D-(10 cm · 10 cm) thin layer chromato- graphy (TLC) on cellulose plates (Merck). First dimension was developed with solvent A (isobutyric acid/concentrated NH4OH/water; 66/1/33; v/v/v); the second dimension was developed with solvent B (concentrated HCl/isopropanol/ water; 18/68/14; v/v/v). When only one dimension was per- formed as in Figure 3, to reduce background, the incubated tRNA was digested with RNase T1, and the fragments were separated on a 20% polyacrylamide gel and the fragment corresponding to the TY-loop was extracted from the gel, eluted, ethanol precipitated and then subjected to nuclease P1 digestion and TLC, as outlined above, except using an acidic solvent for TLC (concentrated HCl/isopropanol/water; 15/70/15; v/v/v). rRNA pseudouridine synthase assay Recombinant pfuCbf5 (2 mg) was incubated with 0.75 pmol unlabeled guide (Pf9) sRNA and 0.05 pmol [a-32P]UTP- labeled-target rRNA as described previously (12). For experi- ments with accessory proteins present, the additional proteins were added at stoichiometric amounts equivalent to 2 mg pfuCbf5. Incubation was at 70 C for 1 h. Resulting RNA was phenol extracted, digested with nuclease P1 for 1 h at 37 C and run on a 1D (20 cm) TLC plate in acidic solvent (concentrated HCl/isopropanol/water; 15/70/15; v/v/v). The results were analyzed by autoradiography. CMC/RT assay for mapping pseudouridine residues Total tRNA was isolated from E.coli cells that were either BL21(DE3), ML100, or ML100 transformed with the pET plasmids containing respectively the cbf5 or pus10 genes. cells (100 ml) were grown to OD 0.4, then protein expression was induced with 0.05 mM IPTG for 3 h at 37 C. Cells were then rapidly transferred from 37 to 50 C and incubated for an additional 2 h. tRNA was isolated according to (21). CMC modiﬁcation was performed on 2 mg of total tRNA as described previously (22). The resulting modiﬁed tRNA samples were then subjected to primer extension using 50 end labeled primers. Primer for ecotRNACys was 50- TGGAGGCGCGTCCGG-30. Primer for ecotRNAPhe was 50-TGGTGCCCGGACTCGG-30. Primer (0.5 pmol) was annealed to tRNA in 50 mM Tris–HCl pH 8.6, 60 mM NaCl and 10 mM DTT for 3 min at 70 C, 5 min at 37 C and at least 2 min at 0 C. Primer extension was performed using 2 ml of the above annealing reaction in a 5 ml reaction mixture containing 50 mM Tris–HCl pH 8.6, 60 mM NaCl, 10 mM DTT and 2.4 mM MgCl2, 330 mM dNTPs and 20 U reverse transcriptase (Promega). Reaction was carried out for 30 min at 37 C. For sequencing reactions, 10 mg of unmodiﬁed wild type total tRNA was used in the same annealing reaction conditions as before, except using 1.5 pmol of labeled primer. Extension was performed in the same buffer as above, except with the addition of 1 mM individual ddNTPs in four different reactions. The resulting fragments were separated on a 15% sequencing acrylamide gel (7 M urea). Results were analyzed by autoradiography. Nucleic Acids Research, 2006, Vol. 34, No. 15 4295 RESULTS Both pfuCbf5 and pfuPus10 can form C55 in tRNA in vitro As described above, bioinformatic searches yielded two can- didate enzymes for U55 modiﬁcation in archaea. pfuCbf5 is a member of the TruB/Pus4/Cbf5 family of enzymes that are responsible for Y55 modiﬁcation in bacteria and eukaryotes. pfuPus10 (previously PsuX) is a member of another family of pseudouridine synthases with unknown speciﬁcity. To test whether these enzymes are tRNA Y55 synthases, we cloned the P.furiosus cbf5 and pus10 genes, expressed and puriﬁed the pfuCbf5 and pfuPus10 proteins and assayed their activity. Both enzymes catalyzed the formation of Y in vitro (Figure 1). More extensive tests were then performed to further analyze the activity of pfuCbf5 and pfuPus10. Both enzymes were found to work under the same reaction conditions, with the optimal activity observed at 1 h at 70 C (Figure 1C–F). pfuPus10 efﬁ- ciently produced 1 mol of Y for each mol of tRNA in the reac- tion, suggesting complete modiﬁcation of a single site in the tRNA. The maximum extent of modiﬁcation observed with pfuCbf5 in this assay was 0.6 mol Y per mole of substrate RNA (Figure 1A, C and E). This limitation could reﬂect the sensitivity of Cbf5 to alterations in the substrate RNA or the requirement of Cbf5 for accessory proteins (see below). We determined the sites of tRNA modiﬁcation for each enzyme by performing a combination of nearest neighbor analysis and mutational analysis. Wild type in vitro tran- scribed pfutRNAAsp was (32P)-radiolabeled with one of the four nucleotides. The RNA was incubated with the protein under the optimal reaction conditions (Figure 1). The modi- ﬁed tRNA was isolated and digested with either RNase T2 or nuclease P1 as indicated (Figure 2C and see Figure 2B). The resulting mononucleotides were analyzed by two- dimensional thin layer chromatography (2D-TLC) followed by autoradiography. For both pfuCbf5 and pfuPus10, Y is only detected when tRNA is either UTP-labeled and digested with nuclease P1, or CTP-labeled and digested with RNase T2 (Figure 2C, panels 1–4 and 9–12 and data not shown). These results indicate that the modiﬁed uridine is 50-adjacent to a cytidine. This limits the modiﬁed uridine to positions 22, 35, 39, 55 and/or 60 [numbered according to yeast tRNAPhe (4)]. To test whether the modiﬁed uridine is formed at posi- tion 55, a U55C mutant substrate was constructed in which U55 was replaced with C55, which cannot be modiﬁed. For pfuCbf5 (Figure 2C, panels 5–6), this mutation completely abolished activity. For pfuPus10 (Figure 2C, panels 13–14), this mutation also dramatically reduced the amount of Y pro- duced. The small amount of Y seen with pfuPus10 may be owing to inefﬁcient modiﬁcation of other sites on the tRNA under the in vitro reaction conditions. These results clearly indicate that both pfuCbf5 and pfuPus10 can speciﬁcally mod- ify U55 to Y55 in vitro. Previously, we identiﬁed U55 modiﬁcation activity in a crude P.furiosus cell extract (11). This extract was active on both a wild-type tRNA substrate and a truncated mini-substrate (miniS) that contains only the TY-arm and the acceptor stem (outlined in Figure 2A) (11). To test the ability of the two candidate proteins to act on this substrate, [a-32P]UTP- radiolabeled transcripts of the miniS were incubated with the pfuCbf5 or pfuPus10 proteins, and analyzed as described