4296 Nucleic Acids Research, 2006, Vol. 34, No. 15 Figure 1. Y formation by pfuCbf5 and pfuPus10 in pfutRNAAsp. (A and B) Increasing molar amounts of pfuCbf5 and pfuPus10 were incubated at 70 C for 60 min. (C and D) 0.12 nmol (5 mg) of pfuCbf5 and 0.10 nmol (5 mg) of pfuPus10 were incubated with T7-transcribed pfutRNAAsp for increasing time intervals at 70 C. (E and F) 0.12 nmol (5 mg) of pfuCbf5 and 0.10 nmol (5 mg) of pfuPus10 were incubated at increasing temperatures for 60 min. After incubation, the tRNA was recovered, hydrolyzed by nuclease P1 and the resulting nucleotides were separated by 2D-TLC chromatography (see Materials and Methods). Mol of Y produced per mol of tRNA was measured after counting the radioactivity in the spots corresponding to UMP and YMP on the chromatogram and taking into account the nucleotide composition of the tRNA substrate. Precision is estimated to be 10%. The results shown are representative of two similar experiments. above. As observed in Figure 2C panel 7, pfuCbf5 was unable to modify any uridine residue in the mini-substrate. In contrast, pfuPus10 was active on the mini-substrate (Figure 2C, panel 15). The binding of pfuCbf5 to H/ACA guide RNAs (for rRNA modiﬁcation) was previously found to depend on the pres- ence of an intact ACA sequence near the 30 terminus of the guide RNA (box ACA) and mutations in this element pre- vented Cbf5 activity toward rRNA substrate (12,13). tRNAs contain a CCA sequence at the 30 end. To determine whether the 30CCA plays an important role in recognition of tRNA by pfuCbf5, we generated a tRNA that lacked the terminal CA (see dashed outline in Figure 2A). As shown in Figure 2C (panels 8 and 16), pfuCbf5 did not modify the tRNA 30CA mutant. At the same time, the 30CA mutation did not affect the ability of pfuPus10 to modify the substrate tRNA. Y55 formation by pfuCbf5 in the absence and presence of accessory proteins While the known tRNA pseudouridylations are introduced by dedicated, single-subunit enzymes, we know that in order to function with guide RNAs to modify rRNA in archaea and eukaryotes, Cbf5 requires accessory proteins: Gar1, L7Ae and Nop10 (12,13). In order to determine whether the accessory proteins could also be important in tRNA U55 modiﬁcation, pseudouridylation assays were performed in the absence and presence of these proteins.