Nucleic Acids Research, 2006, Vol. 34, No. 15 4297 Figure 2. Analysis of pfuCbf5 and pfuPus10 modification of various tRNA substrates. (A) Cloverleaf structure of tRNAAsp of P.furiosus used as the wild-type tRNA substrate in this work. The universal numbering system for nucleotides in tRNA corresponds to that of (4). U55 is indicated. C75 and A76 (missing in 30CA substrates) are indicated by a dashed box. The portion of the tRNA sequence included in the miniS substrate is indicated by the plain box. (B) The expected patterns of nuclease P1 and RNase T2 cleavage in the U55 region of an [a-32P]UTP- and [a-32P]CTP-labeled tRNA, respectively. Nuclease P1 generates 50-phosphate-nucleosides while RNase T2 generates 30phosphate-nucleosides. (C) 2D-TLC analysis of pfuCbf5 and pfuPus10 modification of various tRNA substrates. WT indicates wild-type pfutRNAAsp (panels 1–4, 9–12); U55C indicates U55 replacement mutant (panels 5,6,13,14); miniS indicates mini- substrate (panels 7,15) and 30CA indicates 30 terminal CA deletion (panels 8,16). See text for more details. UTP/CTP/ATP and GTP refer to the (32P)-labeled nucleotide incorporated at transcription. Incubation was for 1 h at 70 C in the presence of 0.12 nmol (5 mg) of pfuCbf5 (panels 1–8) and 0.01 nmol (0.5 mg) of pfuPus10 (panels 9–16). After incubation, the RNA was digested by nuclease P1 or RNase T2 (as indicated in each panel) and the resulting nucleotides were analyzed by 2D-TLC on cellulose plates and autoradiography. Circles in dotted lines show the migration of the canonical nucleotides used as UV markers. As has been shown previously (12) and is demonstrated in Figure 3 (right panel, rRNA), in rRNA pfuCbf5 converts a radiolabeled uridine (targeted by a speciﬁc guide RNA) to Y only in the presence of the three accessory proteins (C+ lane). Uniformly [a-32P]UTP-radiolabeled archaeal pfutRNAAsp was also incubated with pfuCbf5 in the absence (C) and pres- ence (C+) of stoichiometric amounts of the accessory proteins (Figure 3, left panel). After incubation, the tRNA was isol- ated and digested with RNase T1, and the TY-arm fragment was obtained by gel puriﬁcation. The TY-arm fragment con- sists of nucleotides 54–65 and contains three uridines. The isolated fragment was digested with nuclease P1 and the mononucleotides were separated by single dimension TLC. As shown in Figure 3 (left panel), pfuCbf5 produced 0.6 mol of Y per mol of tRNA in the absence of the accessory proteins, and 0.9 mol of Y per mol of tRNA in the presence of the other proteins. The same extent of enhancement ( 50% increase) was observed in two experiments. In order to determine which of the three uridines within the TY arm of the tRNA was modiﬁed in this assay, a mutant pfutRNAAsp in which U55 was replaced by C55 was tested (Figure 3, middle panel, tRNAU55C). No pseudouridylation was observed in the tRNA lacking U55, indicating that the pseudouridylation activity observed is directed speciﬁcally at uridine 55. Our ﬁndings indicate that pfuCbf5 can modify tRNA U55, but not rRNA, in the absence of Gar1, Nop10 and L7Ae. However, the activity of pfuCbf5 toward tRNA U55 is enhanced in the presence of the accessory proteins, suggest- ing that a multi-protein Cbf5 complex may function in Y55 synthesis in P.furiosus. pfuPus10 can efficiently form Y55 in tRNA in E.coli Our results indicate that both pfuCbf5 and pfuPus10 can cata- lyze tRNA Y55 formation in vitro. Unfortunately, because P.furiosus is not currently amenable to genetic analysis, we are unable to unequivocally determine which protein is responsible for U55 modiﬁcation in this organism. Therefore we tested for the ability of the enzymes to modify tRNA U55 in a heterologous system—an E.coli strain harboring a