Cupric ion catalysis in hydrolysis of aminoacyl-tRNA

Schofield, Philip J.; Zamecnik, Paul C.

doi:10.1016/0005-2787(68)90185-8

Cited by 132 publications

(56 citation statements)

References 11 publications

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“…Free amino acid was cleared by ethanol precipitation. Residual copper ions were extracted by chromatography over a column containing 300 l of Chelex 100 resin (Sigma) in 150 mM sodium acetate (pH 5.5) (18). Prior to use in kinetic assays the substrate was ethanolprecipitated, dried at room temperature, and dissolved in sterile H 2 O.…”

Section: Methodsmentioning

confidence: 99%

Crystal Structure of a Human Peptidyl-tRNA Hydrolase Reveals a New Fold and Suggests Basis for a Bifunctional Activity

Pereda

Waas

Jan

et al. 2004

Journal of Biological Chemistry

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Peptidyl-tRNA hydrolase (Pth) activity releases tRNA from the premature translation termination product peptidyl-tRNA. Two different enzymes have been reported to encode such activity, Pth present in bacteria and eukaryotes and Pth2 present in archaea and eukaryotes. Here we report the crystallographic structure of the Homo sapiens Pth2 at a 2.0-Å resolution as well as its catalytic properties. In contrast to the structure of Escherichia coli Pth, H. sapiens Pth2 has an ␣/␤ fold with a four-stranded antiparallel ␤-sheet in its core surrounded by two ␣-helices on each side. This arrangement of secondary structure elements generates a fold not previously reported. Its catalytic efficiency is comparable with that reported for the archaeal Sulfolobus solfataricus Pth2 and higher than that of the bacterial E. coli Pth. Several lines of evidence target the active site to two close loops with highly conserved residues. This active site architecture is unrelated to that of E. coli Pth. In addition, intermolecular contacts in the crystal asymmetric unit cell suggest a likely surface for protein-protein interactions related to the Pth2-mediated apoptosis.During the protein translation process, a significant proportion of the ribosomes that initiate mRNA readout do not reach the stop codon. Peptidyl-tRNA molecules may dissociate from the mRNA template causing a premature end of the process (1, 2). Accumulation of peptidyl-tRNAs reduces the efficiency of translation by sequestering tRNAs and impairing the initiation (3). Prokaryotic and eukaryotic cells show an enzymatic activity that releases the peptidyl moiety from the tRNA, called peptidyl-tRNA hydrolase (Pth), 1 allowing the free tRNA and peptide to be reused in protein synthesis (4, 5). First identified in Escherichia coli, genes encoding Pth have been found in bacteria and eukaryotes but not in archaea. E. coli Pth crystal structure has been solved and exhibits an ␣/␤ fold formed by three layers with a mixed five-strand ␤-sheet at the core. Its catalytic parameters have been measured, and several residues affecting the 5Ј-phosphate and 3Ј-end recognition of the peptidyl-tRNA substrate have been mapped (6, 7).Recently, a Pth-like activity has been identified in Methanocaldococcus jannaschii and characterized in Sulfolobus solfataricus, both archaebacteria (8, 9). The new enzyme called Pth2 is shorter in length and lacks any significant sequence homology to Pth. It coincides with the Pfam data base entry "Uncharacterized Protein Family 0099" (UPF0099) found in archaea and eukaryotes but not in bacterial genomes. Pth2 lacks any detectable homology to solved three-dimensional structures. S. solfataricus Pth2 displays a greater catalytic efficiency toward E. coli diacetyl-lysyl-tRNA Lys than E. coli Pth (9, 6). In addition, both enzymes differ in their mode of recognition of several tRNA features, like the 1-72-base pair mismatch in E. coli tRNA fMet or the presence of a phosphorylated 5Ј-end.Human Pth2, also called Bcl-2 inhibitor of transcription 1 (Bit1), possesses an ...

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Section: Methodsmentioning

confidence: 99%

Crystal Structure of a Human Peptidyl-tRNA Hydrolase Reveals a New Fold and Suggests Basis for a Bifunctional Activity

Pereda

Waas

Jan

et al. 2004

Journal of Biological Chemistry

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“…Acetylation of the aminoacyl-tRNA was achieved on ice for 1 h by the addition of 0.8 ml of 4 M potassium acetate, pH 5.0, and five aliquots of 20 l of acetic anhydride at intervals of 12 min (13). After ethanol precipitation and centrifugation, the pellets were dissolved in 0.2 ml of DEPC-treated water, and the degree of acetylation was determined by the cupric reaction (14). The amount of substrate available for the Pth reaction was determined by spotting a sample onto a 3-mm filter disk (Whatman).…”

Section: Methodsmentioning

confidence: 99%

Orthologs of a novel archaeal and of the bacterial peptidyl–tRNA hydrolase are nonessential in yeast

Rosas-Sandoval

Ambrogelly

Rinehart

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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Peptidyl-tRNA hydrolase (encoded by pth) is an essential enzyme in all bacteria, where it releases tRNA from the premature translation termination product peptidyl-tRNA. Archaeal genomes lack a recognizable peptidyl-tRNA hydrolase (Pth) ortholog, although it is present in most eukaryotes. However, we detected Pth-like activity in extracts of the archaeon Methanocaldococcus jannaschii. The uncharacterized MJ0051 ORF was shown to correspond to a protein with Pth activity. Heterologously expressed MJ0051 enzyme catalyzed in vitro the cleavage of the Pth substrates diacetyl-[ 14 C]lysyl-tRNA and acetyl-[ 14 C]phenylalanyl-tRNA. On transformation of an Escherichia coli pth ts mutant, the MJ0051 gene (named pth2) rescued the temperature-sensitive phenotype of the strain. Analysis of known genomes revealed the presence of highly conserved orthologs of the archaeal pth2 gene in all archaea and eukaryotes but not in bacteria. The phylogeny of pth2 homologs suggests that the gene has been vertically inherited throughout the archaeal and eukaryal domains. Deletions in Saccharomyces cerevisiae of the pth2 (YBL057c) or pth (YHR189w) orthologs were viable, as was the double deletion strain, implying that the canonical Pth and Pth2 enzymes are not essential for yeast viability. P eptidyl-tRNA hydrolase (Pth) is an essential enzyme in bacteria (1-3). It cleaves peptidyl-tRNAs, premature protein synthesis products that dissociate from the ribosome, and allows the discharged tRNAs to reengage in protein synthesis. In mutants with limited Pth activity, peptidyl-tRNAs accumulate in the cell and reduce the availability of essential acylatable tRNAs below the limit compatible with protein synthesis; thus cell growth is impaired. However, the possibility that peptidyl-tRNA per se is toxic has not been ruled out. A significant proportion of the ribosomes that initiate mRNA translation interrupt protein synthesis before reaching the stop codon (4). These events of abortive translation may result in peptidyl-tRNA dissociation from the ribosome (5), forming the natural substrates for Pth. There are at least two documented instances of peptidyl-tRNA accumulation and growth inhibition in Escherichia coli mutants partly defective in Pth activity: minigene expression and overproduction of a nonfunctional protein. In each case, accumulation of peptidyl-tRNA occurs corresponding to the last sense codon, implying defects in translation termination (6-8).Pth activity is ubiquitous. Orthologs of the E. coli pth gene have been identified in all known bacterial and some eukaryotic genomes (e.g., yeast, maize, mice, and human), and Pth activities were demonstrated in yeast long ago (9-11). Furthermore, the bacterial pth ortholog YHR189w of Saccharomyces cerevisiae complements a temperature-sensitive E. coli strain harboring a pth ts mutant, although YHR189w is not essential for yeast viability (3). Biochemical studies with S. cerevisiae extracts revealed the presence of at least two types of Pth activities, distinguished by the different efficiency o...

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“…After washing the column with 300 mM NaCl in buffer I, aminoacylated and formylated tRNA was eluted with 700 mM NaCl in buffer I and desalted on Biogel P6. Aminoacylation and formylation were assayed by acid precipitation in the presence of CuSO, as described (Schofield and Zamecnik, 1968).…”

Section: Methodsmentioning

confidence: 99%

Identification and Purification of Translation Initiation Factor 2 (IF2) from Thermus thermophilus

Vornlocher

Scheible

Faulhammer

et al. 1997

European Journal of Biochemistry

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Translation initiation factor 2 (IF2) is one of three protein factors required for initiation of protein synthesis in eubacteria. The protein is responsible for binding the initiator tRNA to the ribosomal P site. IF2 is a member of the GTP/GDP-binding protein superfamily. In the extreme thermophilic bacterium Themus rhermophilus, IF2 was identified as a 66-kDa protein by affinity labeling and immunoblotting. The protein was purified to homogeneity. The specific activity indicates a stoichiometric IF2-mediated binding of formylmethionine-tRNA to 70s ribosomes. The N-terminal amino acid sequences of the intact protein and of two proteolytic fragments of 25 kDa and 40 kDa were determined. Comparison with other bacterial IF2 sequences indicates a similar domain architecture in all bacterial IF2 proteins.

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Cupric ion catalysis in hydrolysis of aminoacyl-tRNA

Cited by 132 publications

References 11 publications

Crystal Structure of a Human Peptidyl-tRNA Hydrolase Reveals a New Fold and Suggests Basis for a Bifunctional Activity

Crystal Structure of a Human Peptidyl-tRNA Hydrolase Reveals a New Fold and Suggests Basis for a Bifunctional Activity

Orthologs of a novel archaeal and of the bacterial peptidyl–tRNA hydrolase are nonessential in yeast

Identification and Purification of Translation Initiation Factor 2 (IF2) from Thermus thermophilus

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