Figure 4. Detection of Y in E.coli tRNACys and tRNAPhe. (A) CMC/RT analysis of tRNACys of E.coli. tRNA was isolated from wild-type (wt), truB (KO) or truB transformed with a plasmid containing the pfu pus10 gene (+Pus10) E.coli and CMC modified. Primer extension was performed as described in Materials and Methods with a primer complementary to ecotRNACys, nucleotides 61–76. The arrow indicates a strong stop at Y55. (B) CMC/RT analysis of ecotRNAPhe as described above using a primer complementary to nucleotides 61–76 of ecotRNAPhe. enhanced in the presence of the accessory proteins. Thus, in both cases the relevant enzyme may be a multi-protein complex. The dual function of archaeal Cbf5 in tRNA and rRNA modiﬁcation may account for structural similarities that have been noted between components of these pseudouridyla- tion reactions from different systems. The X-ray crystal struc- tures of bacterial TruB and archaeal Cbf5 are remarkably similar (27–31). Moreover, the structure of the regions of the substrate RNAs recognized and modiﬁed by TruB and Cbf5, the TY-arm of tRNA and guide RNA/rRNA duplex respectively, are strikingly comparable (29,32–34). Indeed this similarity was recently used to model the interaction between pfuCbf5 and the guide RNA/rRNA complex (29). Our ﬁndings suggest that pfuCbf5 may also recognize a com- mon feature of tRNAs and H/ACA guide RNAs outside the area of modiﬁcation—the CCA and ACA at the 30 ends of Nucleic Acids Research, 2006, Vol. 34, No. 15 4299 tRNAs and H/ACA RNAs, respectively [Figure 3 and (12)]. These conserved structural features between tRNA and rRNA modiﬁcation systems may be a consequence of the dual function of Cbf5 in both processes in archaea. Alternat- ively, the observed activity of Cbf5 toward tRNA may reﬂect general conservation between Y synthases. Y55 synthases in the three domains of life Modiﬁcation of U55 in tRNA to Y55 occurs in all three domains of life. In bacteria, TruB is responsible for this modi- ﬁcation (10,35). In eukaryotes (with few exceptions including Drosophila melanogaster and Caenorhabditis elegans) the tRNA Y55 synthase is Pus4 (15). The results presented here implicate Cbf5 or Pus10 (or both) as the enzyme responsible for this modiﬁcation in archaea. Cbf5 also functions in H/ACA RNA-guided modiﬁcation of rRNA in archaea (12,13). rRNA modiﬁcations are also introduced by Cbf5 (by an RNA-guided mechanism) in eukaryotes (14). Pseudo- uridylation of rRNA is carried out by a set of site-speciﬁc enzymes in bacteria (36). Pus10 is more distantly related to these other Y synthases. The overall domain organization of Pus10 differs signiﬁc- antly from the other Y55 synthases (Figure 5). Sequence ana- lysis of Pus10 reveals the presence of several distinct domains (15). CXXC Zn binding motifs are found in the N-terminal region of Pus10 proteins (15). A similar motif is present in Pus1, a distinct tRNA Y synthase in S.cerevisiae, which contains one atom of Zn essential for the native con- formation and tRNA recognition (37). A THUMP domain (present in some THioUridine synthases, Methylases and Pseudouridine synthases) is located adjacent to the CXXC Zn binding motifs of Pus10 (38). This characteristic motif is proposed to be involved in RNA binding (38). A recent study indicates that the THUMP motif alone cannot bind RNA, but facilitates the interaction of the catalytic domain of the archaeal Trm-G10 methyltransferase enzyme with tRNA sub- strates (39). The Pus10-related proteins of Drosophila and Caenorhabditis lack this THUMP domain (38). The catalytic domain of Pus10 appears to be found in the C-terminal region of the protein (15). TruB and Cbf5 (but not Pus4 of S.cerevisiae) contain a characteristic PUA domain (so called because it is present in PseudoUridine synthases and Archaeosine transglycosy- lase) within the C-termini of the proteins (40,41) (Figure 5). In archaeal Cbf5, the PUA domain plays a crucial role in binding H/ACA guide RNAs [(28), D. Baker, R. M. Terns and M. P. Terns, unpublished data]. This RNA binding depends on the presence of an ACA sequence at the 30 end of the guide RNA (28,29). We have found that archaeal Cbf5 also requires an intact 30 terminal CCA sequence for activity toward tRNA (Figure 2). Evolutionary considerations The observations made in this study may provide some insight on the evolutionary origin of the present-day RNA Y syn- thases, especially the tRNA Y55 synthases. The ﬁnding that in archaea Cbf5 may function in tRNA U55 pseudouridylation as well as RNA-guided pseudouridylation of rRNA supports the idea that Cbf5 is a direct descendant of a primordial TruB/Pus4-like tRNA Y synthase. This primordial Y synthase