4294 Nucleic Acids Research, 2006, Vol. 34, No. 15 eukaryotes and archaea members of the Cbf5 sub-family modify multiple speciﬁc sites by a mechanism that depends on guide RNAs and a set of essential accessory proteins (14). Thus in most eukaryotes, the paralogous proteins Pus4 and Cbf5 function in tRNA U55 and rRNA pseudouridyla- tion, respectively. The functional complex is comprised of Cbf5 (the enzyme) plus Nop10, Gar1 and L7Ae (essential accessory proteins) and an H/ACA guide RNA (12,13). The guide RNA mediates substrate recognition by basepairing with nucleotides surrounding the target uridine. Multiple guide RNAs provide for pseudouridylation of numerous individual rRNA sites. Once recruited to the substrate rRNA by the guide RNA, the complex catalyzes formation of Y at the targeted uridine residue (12). There is no existing evidence that Cbf5 functions in the absence of a guide RNA and accessory proteins, or that Cbf5 is involved in pseudour- idylation of tRNA. The second candidate Y55 synthase that we tested has been called PsuX (15). The psuX gene (hereafter the pus10 gene) was identiﬁed by sequence similarity to an internal region (catalytic domain; residues G66–A271) of the Euglena gracilis Cbf5 protein (15). Pus10 (PF1139) belongs to a different Y synthase family than TruB, Pus4, and Cbf5 [COG 1258 for Pus10, and COG 0130 for the three other Y synthases (8)] and its function and speciﬁcity are unknown. In this work, the activities of recombinantly expressed and puriﬁed pfuCbf5 and pfuPus10 enzymes were characterized and compared in in vitro assays. The results indicate that both enzymes are capable of selective modiﬁcation of tRNA U55 in vitro under similar reaction conditions. We found that pfuPus10 can pseudouridylate a truncated tRNA substrate (miniS) and a tRNA lacking the 30CCA, but that pfuCbf5 functions only on the full-length tRNA substrate including the 30CCA end. Moreover, pfuPus10 efﬁciently complements tRNA Y55 formation in an E.coli truB knockout strain. These results suggest two novel and interesting candidates for the pseudouridylation of tRNA U55 in archaea. MATERIALS AND METHODS Strains and plasmids To construct the ML100 strain (BL21(DE3)truB), the truB2422::mini-Tn10Cm allele from strain GOB113 (16) was transferred by P1 transduction into strain BL21(DE3). Transductants were selected on LB medium supplemented with chloramphenicol (30 mg/ml). The GOB113 strain (Hfr P4X sdr+ truB2422::mini-Tn10Cm) carrying a mutation in the truB gene making this strain defective for TruB activity was kindly provided by Michael Wikstro¨m (University of Umea˚, Sweden). The construction of the pET21d plasmid allowing T7-expression in E.coli of the P.furiosus Cbf5 protein (pfuCbf5; ORF ¼ PF1785) and puriﬁcation of the recombinant protein was described previously (12). The plasmid pML156 allowing in vitro transcription of P.furiosus tRNAAsp by T7 RNA polymerase is a pUC18 derivate with the fragment containing the P.furiosus tRNAAsp gene downstream of a T7 promoter cloned in its BamHI/ HindIII sites. This plasmid was kindly provided by Sylvie Auxilien (CNRS, Gif-sur-Yvette, France). The corresponding P.furiosus tRNAAsp(U55C) mutant was obtained with the QuickChange site-directed mutagenesis kit (Stratagene) using pML156 as a template. The oligonucleotides used were 50-CGACCCGGGTCCAAATCCCGG-30 and 50-CCGGGAT- TTGGACCCGGGTCG-30. The sequence of the mutant was veriﬁed. Cloning the P.furiosus pus10 gene, expression and purification of the corresponding pfuPus10 protein The gene encoding P.furiosus Pus10 (ORF ¼ PF1139), was ampliﬁed by PCR from genomic DNA and cloned into the NdeI/XhoI sites of pET28b. The primers used in this PCR were 50-GTCATATGATACTTGAAAAAGCCA GAG-30 and 50-GTCTCGAGTCAATTATCTCC CTCAACATCG-30. The resulting plasmid (pET28b-pus10) allowed T7-expression in E.coli [strain Rosetta(DE3)] of the P.furiosus Pus10 pro- tein (pfuPus10) bearing an N-terminal His-tag. The trans- formed cells were grown to an optical density at 660 nm of 0.5, and the expression of the protein was induced with 1 mM isopropylthio-b-D-galactoside (IPTG) for 3 h at 37 C. Cells were then harvested and resuspended in buffer A (20 mM Na phosphate pH 7.0, 1 M NaCl) and lyzed by a 30 min sonication at 4 C using a Vibracell 75 041 sonicator (40% amplitude). The lysate was cleared by centrifugation (20 000 g for 30 min) and was applied to a column of Chelating Sepharose Fast Flow (1 · 30 cm; Amersham Biosciences) charged with Ni2+. The column was washed with buffer A and adsorbed material was eluted with a linear gradient (from 0 to 1 M) of imidazole in buffer A. Eluted fractions were analyzed by SDS–PAGE. Fractions containing the pfuPus10 protein were pooled, concentrated to 3 mg/ml in buffer containing 1 M NaCl and 200 mM imidazole and kept at 80 C until used. Preparation of radiolabeled tRNA substrates The general procedure for generating (32P) radiolabeled tRNA transcripts as substrates for enzymatic reactions is based on the method described previously (17). Radioactive (32P) in vitro transcripts were prepared as described else- where (18) using MvaI (for generating tRNAAsp substrate) or NarI (for generating tRNAAsp-30CA substrate) digested plasmids as templates. [a-32P]UTP, [a-32P]CTP, [a-32P]ATP and [a-32P]GTP (400 Ci/mmol) were from ICN Biomedicals and T7 RNA polymerase was from Roche Diagnostics. Radioactive transcripts were puriﬁed by 10% polyacrylamide gel electrophoresis. The radioactive band corresponding to full-length transcript was eluted from the gel with water and ﬁnally precipitated by ethanol in the presence of 0.3 M Na acetate pH 5. The P.furiosus tRNAAsp minisubstrate (TY- loop & stem—see Figure 2A) was prepared by in vitro tran- scription of a single stranded DNA template (minus stand), following the procedure described previously (19). tRNA pseudouridine synthase assay Puriﬁed recombinant pfuCbf5 (5 mg or 2 mg) or pfuPus10 (5 mg or 0.5 mg) was incubated in a 400 ml reaction mixture with the in vitro transcribed (32P)-labeled tRNA for 1 h at 0 C in buffer B (20 mM HEPES pH 7.0, 500 mM KCl and 1.5 mM MgCl2). The reaction was terminated by the addition of an equal volume of phenol and the RNA recovered by ethanol precipitation. The modiﬁed tRNA was then treated