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Orthologs of a Novel Archaeal and of the Bacterial Peptidyl–Trna Hydrolase Are Nonessential in Yeast

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Orthologs of a Novel Archaeal and of the Bacterial Peptidyl–Trna Hydrolase Are Nonessential in Yeast Orthologs of a novel archaeal and of the bacterial peptidyl–tRNA hydrolase are nonessential in yeast Guillermina Rosas-Sandoval†‡, Alexandre Ambrogelly‡§, Jesse Rinehart§, David Wei§, L. Rogelio Cruz-Vera†, David E. Graham¶, Karl O. Stetterʈ, Gabriel Guarneros†, and Dieter So¨ ll§††‡‡ Departments of §Molecular Biophysics and Biochemistry and ††Chemistry, Yale University, New Haven, CT 06520-8114; †Departamento de Gene´tica y Biologı´aMolecular, Centro de Investigacio´n y de Estudios Avanzados del Instituto Polite´cnico Nacional, A. Postal 14-740, Mexico City, Mexico 7000; ¶Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0308; and ʈLehrstuhl fu¨r Mikrobiologie und Archaeenzentrum, Universita¨t Regensburg, D-8400 Regensburg, Germany Contributed by Dieter So¨ll, October 30, 2002 Peptidyl–tRNA hydrolase (encoded by pth) is an essential enzyme Curiously, the known archaeal genomes do not contain any pth in all bacteria, where it releases tRNA from the premature trans- orthologs, raising the question whether Pth activity is dispens- lation termination product peptidyl–tRNA. Archaeal genomes lack able for metabolism in these organisms or whether the activity a recognizable peptidyl–tRNA hydrolase (Pth) ortholog, although it is provided by a nonorthologous enzyme. Therefore we searched is present in most eukaryotes. However, we detected Pth-like for a Pth activity in Methanocaldococcus jannaschii. activity in extracts of the archaeon Methanocaldococcus jannaschii. The uncharacterized MJ0051 ORF was shown to correspond to a Materials and Methods protein with Pth activity. Heterologously expressed MJ0051 en- General. E. coli lysyl–tRNA synthetase was prepared as described zyme catalyzed in vitro the cleavage of the Pth substrates diacetyl- (12). Unfractionated E. coli RNA was purchased from Roche [14C]lysyl–tRNA and acetyl-[14C]phenylalanyl–tRNA. On transfor- Applied Science. [14C]Lys (318 mCi͞mmol) and [14C]Phe (469 mation of an Escherichia coli pthts mutant, the MJ0051 gene mCi͞mmol) were from Amersham Pharmacia Biosciences. (named pth2) rescued the temperature-sensitive phenotype of the RNase inhibitor, Complete protease inhibitor mixture tablets, strain. Analysis of known genomes revealed the presence of highly and Expand High Fidelity PCR System were purchased from conserved orthologs of the archaeal pth2 gene in all archaea and Roche Diagnostics. E. coli BL21-CodonPlus(DE3)-RIL was eukaryotes but not in bacteria. The phylogeny of pth2 homologs from Stratagene. Ni-NTA agarose was from Qiagen (Chats- suggests that the gene has been vertically inherited throughout worth, CA) and the TOPO TA cloning kit (pCR II-Topo Vector) the archaeal and eukaryal domains. Deletions in Saccharomyces from Invitrogen. cerevisiae of the pth2 (YBL057c) or pth (YHR189w) orthologs were viable, as was the double deletion strain, implying that the canon- Purification of Pth Activity from M. jannaschii Cells. Due to the low ical Pth and Pth2 enzymes are not essential for yeast viability. concentration of a very active protein in M. jannaschii or its high sensitivity to proteolytic cleavage, the purification of the Pth activity included pooling of fractions from different purification eptidyl–tRNA hydrolase (Pth) is an essential enzyme in attempts. A general purification scheme is indicated below. bacteria (1–3). It cleaves peptidyl–tRNAs, premature protein P Frozen M. jannaschii cells (700 g) were resuspended in 500 ml of synthesis products that dissociate from the ribosome, and allows ⅐ ͞ ͞ buffer A (50 mM Tris HCl, pH 8.0 10 mM MgCl2 10 mM the discharged tRNAs to reengage in protein synthesis. In 2-mercaptoethanol͞2 mM benzamidine-HCl͞two Complete mutants with limited Pth activity, peptidyl–tRNAs accumulate in protease inhibitor mixture tablets͞10% glycerol), sonicated and the cell and reduce the availability of essential acylatable tRNAs centrifuged at 100,000 ϫ g for1hat4°C. This cell-free extract below the limit compatible with protein synthesis; thus cell (S100) was dialyzed in buffer A and then applied to a DE52 growth is impaired. However, the possibility that peptidyl–tRNA (Whatman) column (35 cm ϫ 20 cm2), preequilibrated with per se is toxic has not been ruled out. A significant proportion of buffer A. The activity eluted in the flowthrough of the column. the ribosomes that initiate mRNA translation interrupt protein The active fractions were precipitated by ammonium sulfate synthesis before reaching the stop codon (4). These events of (80% wt͞vol), resuspended, and dialyzed into buffer B [20 mM abortive translation may result in peptidyl–tRNA dissociation potassium phosphate, pH 6.5͞10 mM 2-mercaptoethanol͞2mM from the ribosome (5), forming the natural substrates for Pth. benzamidine⅐HCl͞one Complete protease inhibitor mixture tab- There are at least two documented instances of peptidyl–tRNA let (per 50 ml)͞10% glycerol] and applied to a P11 (Whatman) accumulation and growth inhibition in Escherichia coli mutants column (20 cm ϫ 20 cm2) preequilibrated with the same buffer. partly defective in Pth activity: minigene expression and over- The fractions containing the activity were pooled, concentrated production of a nonfunctional protein. In each case, accumula- with polyethylene glycol 15,000–20,000 Mr, and dialyzed against tion of peptidyl–tRNA occurs corresponding to the last sense buffer B. The dialyzed fractions were applied to a hydroxyl codon, implying defects in translation termination (6–8). apatite column and eluted with a linear 20–500 mM potassium Pth activity is ubiquitous. Orthologs of the E. coli pth gene phosphate (pH 6.5) gradient. The active fractions were pooled, have been identified in all known bacterial and some eukaryotic concentrated, dialyzed into fresh buffer B, and reapplied to the genomes (e.g., yeast, maize, mice, and human), and Pth activities column. After elution with a second linear gradient of 20–375 were demonstrated in yeast long ago (9–11). Furthermore, the mM phosphate, the active fractions were pooled, concentrated, bacterial pth ortholog YHR189w of Saccharomyces cerevisiae and dialyzed against buffer C (50 mM sodium phosphate, complements a temperature-sensitive E. coli strain harboring a pH 7.0͞0.3 M NaCl͞10% glycerol). The sample was then applied pthts mutant, although YHR189w is not essential for yeast to a 320 ml Superdex 75PG column (Amersham Pharmacia viability (3). Biochemical studies with S. cerevisiae extracts Biosciences). The active protein(s) eluted after the 25-kDa revealed the presence of at least two types of Pth activities, distinguished by the different efficiency of cleaving various substrates (9). A Pth activity in yeast that acts only on acetyl– Abbreviations: IPTG, isopropyl-␤-D-thiogalactopyranoside; Pth, peptidyl–tRNA hydrolase. Phe–tRNA has also been reported (11). Thus, there may be ‡G.R.-S. and A.A. contributed equally to this work. BIOCHEMISTRY several Pth-like activities in yeast. ‡‡To whom correspondence should be addressed. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.222659199 PNAS ͉ December 24, 2002 ͉ vol. 99 ͉ no. 26 ͉ 16707–16712 Downloaded by guest on September 25, 2021 standard (chymotrypsinogen A) and just before the 13.7-kDa water. Acetylation of the aminoacyl–tRNA was achieved on ice standard (ribonuclease A). for1hbytheaddition of 0.8 ml of 4 M potassium acetate, pH 5.0, and five aliquots of 20 ␮l of acetic anhydride at intervals of Peptide Sequencing. The peak sample was concentrated in poly- 12 min (13). After ethanol precipitation and centrifugation, the ethylene glycol to 30 ␮l, mixed with 4ϫ SDS loading buffer, pellets were dissolved in 0.2 ml of DEPC-treated water, and the heated to 90°C for 5 min, and applied to 12% SDS͞PAGE gel degree of acetylation was determined by the cupric reaction (14). (20 ϫ 20 cm). The gel was stained with Coomassie brilliant blue. The amount of substrate available for the Pth reaction was A gel piece containing a 27-kDa protein was analyzed by determined by spotting a sample onto a 3-mm filter disk (What- matrix-assisted laser desorption ionization–time of flight anal- man). The filters were then washed three times for 10–15 min in ysis by the Procter & Gamble Pharmaceuticals Structural Biol- cold 10% (wt͞vol) trichloroacetic acid supplemented with 3% ogy Research Group. Eight peptides from a tryptic digest could (wt͞vol) casamino acids, rinsed in cold 95% ethanol, and dried. be identified covering 62% of M. jannaschii MJ0051 protein The radioactivity on the filters was measured in a scintillation (MVVVIR, AVDEWLR, MKMVVVIR, ELIDIYNK, IDKIT- counter. GHLK, SEGLPCSIIR, VNSEKELIDIYNK, DAGHTQ- LEPGTLTAVAIGPEK). Four other hypothetical M. jannaschii Pth Assay and Verification of tRNA Integrity After the Pth Reaction. proteins were also detected in the sample. Reactions were initiated by the addition of 150 ␮l of a pre- warmed (37°C for 5 min) substrate mixture (230 mM Tris⅐HCl, Cloning of the M. jannaschi MJ0051 Gene. ͞ ͞ ͞ Genomic DNA was pH 7.2 185 mM NH4Cl 0.70 mM sodium azide either 227 prepared from M. jannaschii cells by standard methods. The ␮M-667 ␮M diacetyl-[14C]Lys–tRNA or acetyl-[14C]Phe–tRNA) Ј oligonucleotide primers were MJ0051NDE (5 -GAGGTTTAcatat- to 300 ␮l of a reaction mixture [10 mM Tris⅐HCl, pH 7.2͞20 mM gAAGATGGT-TGTAGTAA-3Ј) and MJ0051XHO (5Ј-AATT- ͞ ͞ ͞ Ј NH4Cl 10 mM magnesium acetate 60 units of RNase inhibitor TActcgagTAAAAGTTTTAAATGTCC-3 ). The lowercase let- either M. jannaschii S-100 extract (100 ␮g), M. jannaschii Pth- ters indicate the NdeI and XhoI restriction sites; the translation His-6 (13, 26, and 187 nM) or E. coli Pth (13 and 187 nM)]. The initiation site is shown in bold and italics. ORF MJ0051 was reaction was performed at 37°C. Aliquots (75 ␮l) were taken at amplified by PCR by using the primers above and the Expand High Ϸ different time points and spotted onto a 3-mm filter disk. The Fidelity PCR System. The PCR product of the expected size ( 345 filters were washed, dried, and counted as described above.
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