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Umesh Varshney - One of the best experts on this subject based on the ideXlab platform.
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Mini-Review Peptidyl-tRNA hydrolase and its critical role in protein biosynthesis
2020Co-Authors: Umesh VarshneyAbstract:Peptidyl-tRNA hydrolase (Pth) releases tRNA from Peptidyl-tRNA by cleaving the ester bond between the peptide and the tRNA. Genetic analyses using Escherichia coli harbouring temperature-sensitive Pth have identified a number of translation factors involved in Peptidyl-tRNA release. Accumulation of Peptidyl-tRNA in the cells leads to depletion of aminoacyl-tRNA pools and halts protein biosynthesis. Thus, it is vital for cells to maintain Pth activity to deal with the pollution of Peptidyl-tRNAs generated during the initiation, elongation and termination steps of protein biosynthesis. Interestingly, while eubacteria possess a single class of Peptidyl-tRNA hydrolase, eukaryotes possess several such activities, making Pth a potential drug target to control eubacterial infections. This review discusses the aspects of Pth that relate to its history and biochemistry and its physiological connections with various cellular factors.
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Structural plasticity and enzyme action: crystal structures of mycobacterium tuberculosis Peptidyl-tRNA hydrolase.
Journal of Molecular Biology, 2007Co-Authors: M. Selvaraj, N. S. Singh, Umesh Varshney, R. Sangeetha, M. VijayanAbstract:Peptidyl-tRNA hydrolase cleaves the ester bond between tRNA and the attached peptide in Peptidyl-tRNA in order to avoid the toxicity resulting from its accumulation and to free the tRNA available for further rounds in protein synthesis. The structure of the enzyme from Mycobacteritan tuberculosis has been determined in three crystal forms. This structure and the structure of the enzyme frorn Escherichia coli in its crystal differ substantially on account of the binding of the C terminus of the E. coli enzyme to the peptide-binding site of a neighboring molecule in the crystal. A detailed examination of this difference led to an elucidation of the plasticity of the binding site of the enzyme. The peptide-binding site of the enzyme is a cleft between the body, of the molecule and a polypepticle Y stretch involving a loop and a helix. This stretch is in the open conformation when the enzyme is in the free state as in the crystals of M. tuberculosis Peptidyl-tRNA hydrolase. Furthermore, there is no physical continuity between the tRNA and the peptide-binding sites. The molecule in the E. coli crystal mimics the peptide-bound enzyme molecule. The peptide stretch referred to earlier now closes on the bound peptide. Concurrently, a channel connecting the tRNA and the peptide-binding site opens primarily through the concerted movement of two residues. Thus, the crystal structure of M. tuberculosis Peptidyl-tRNA hydrolase when compared with the crystal structure of the E. coli enzyme, leads to a model of structural changes associated with enzyme action on the basis of the plasticity of the molecule. (c) 2007 Elsevier Ltd. All rights reserved.
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Cloning, expression, purification, crystallization and preliminary X-ray analysis of Peptidyl-tRNA hydrolase from Mycobacterium tuberculosis.
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2006Co-Authors: M. Selvaraj, N. S. Singh, Umesh Varshney, R. Sangeetha, M. VijayanAbstract:Peptidyl-tRNA hydrolase catalyses the cleavage of the ester link between the peptide and the tRNA in Peptidyl-tRNAs that, for various reasons, have dropped off the translating ribosomes. This enzyme from Mycobacterium tuberculosis has been crystallized in three related but distinct forms: $P2_12_12_1$, unit-cell parameters a = 36.30, b = 61.85, c = 73.97 \AA , $P2_1$, a = 35.83, b = 73.79, c = 59.79 \AA ,$_\beta$ = 92.3, and $P2_12_12_1$, a = 35.84, b = 57.06, c = 72.59 \AA . X-ray data have been collected from all three forms.
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Cloning, expression, purification, crystallization and preliminary X-ray analysis of Peptidyl-tRNA hydrolase from Mycobacterium tuberculosis.
Acta crystallographica. Section F Structural biology and crystallization communications, 2006Co-Authors: M. Selvaraj, N. S. Singh, Umesh Varshney, R. Sangeetha, M. VijayanAbstract:Peptidyl-tRNA hydrolase catalyses the cleavage of the ester link between the peptide and the tRNA in Peptidyl-tRNAs that, for various reasons, have dropped off the translating ribosomes. This enzyme from Mycobacterium tuberculosis has been crystallized in three related but distinct forms: P2(1)2(1)2(1), unit-cell parameters a = 36.30, b = 61.85, c = 73.97 A, P2(1), a = 35.83, b = 73.79, c = 59.79 A, beta = 92.3 degrees , and P2(1)2(1)2(1), a = 35.84, b = 57.06, c = 72.59 A. X-ray data have been collected from all three forms.
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Peptidyl-tRNA hydrolase and its critical role in protein biosynthesis.
Microbiology, 2006Co-Authors: Umesh VarshneyAbstract:Peptidyl-tRNA hydrolase (Pth) releases tRNA from Peptidyl-tRNA by cleaving the ester bond between the peptide and the tRNA. Genetic analyses using Escherichia coli harbouring temperature-sensitive Pth have identified a number of translation factors involved in Peptidyl-tRNA release. Accumulation of Peptidyl-tRNA in the cells leads to depletion of aminoacyl-tRNA pools and halts protein biosynthesis. Thus, it is vital for cells to maintain Pth activity to deal with the pollution of Peptidyl-tRNAs generated during the initiation, elongation and termination steps of protein biosynthesis. Interestingly, while eubacteria possess a single class of Peptidyl-tRNA hydrolase, eukaryotes possess several such activities, making Pth a potential drug target to control eubacterial infections. This review discusses the aspects of Pth that relate to its history and biochemistry and its physiological connections with various cellular factors.
Robert L. Mcfeeters - One of the best experts on this subject based on the ideXlab platform.
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Recent Antimicrobial Developments Targeting Peptidyl-tRNA Hydrolases
2020Co-Authors: Robert L. McfeetersAbstract:Peptidyl-tRNA hydrolases (Pths) are essential enzymes found in bacteria, archaea, and eukaryotes. Pths cleave the peptide:tRNA ester bond of Peptidyl-tRNAs generated from premature termination of protein synthesis and the expression of short ORFs or minigenes. Accumulation of Peptidyl-tRNAs is toxic presumably due to impaired translational initiation or slowed protein synthesis caused by specific tRNA starvation. Pth activity is thus vital for cells to deal with the build-up of Peptidyl-tRNAs. Bacterial genomes encode a highly conserved Peptidyl-tRNA hydrolase enzyme, Pth1. Amino acid sequence homology is high across all species and active site residues are strictly conserved. Structures of Pth1 have been solved for several species and catalytically important residues identified. Studies with mini- and partial-substrates have contributed to understanding of substrate recognition. However, the mechanism of action and ability to distinguish between aminoacyl- and Peptidyl-tRNAs are not fully understood.
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Inhibition of essential bacterial Peptidyl-tRNA hydrolase activity by tropical plant extracts.
Natural Product Communications, 2020Co-Authors: Hana Mcfeeters, Luis R. Cruz-vera, Morgan J. Gilbert, Rachel M. Thompson, William N. Setzer, Robert L. McfeetersAbstract:: Peptidyl-tRNA Hydrolase (Pth) is a highly conserved, essential enzyme in bacteria. It removes the peptide portion from Peptidyl-tRNA, returning free tRNAs to participate in translation. Build-up of Peptidyl-tRNAs is toxic and defects in Pth function result in cell death. Herein we use in vitro activity of recombinant E. coli Pth to screen tropical plant extracts for inhibition. Multiple extracts were found to have inhibitory activity with some exhibiting different inhibitory effects depending on extraction conditions. IC50 values ranged from 0.02 to > 53.8 microg of extract per 1 unit of Pth, holding promise for in vivo screening. The inhibitory components in these extracts may serve as lead compounds for development of novel antibacterials.
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Expression, purification, and buffer solubility optimization of the putative human Peptidyl-tRNA hydrolase PTRHD1.
Protein Expression and Purification, 2016Co-Authors: Geordan L. Burks, Hana Mcfeeters, Robert L. McfeetersAbstract:Performing the essential function of recycling Peptidyl-tRNAs, Peptidyl-tRNA hydrolases are ubiquitous in all domains of life. The multicomponent eukaryotic Pth system differs greatly from the bacterial system composed predominantly of a single Pth1 enzyme. While bacterial Pth1s are structurally well characterized and promising new targets for antibiotic development, eukaryotic Pths are largely understudied. From amino acid sequence alignment and secondary structure predictions, the human gene product PTRHD1 was classified as a eukaryotic Pth. Herein, we report cloning, recombinant bacterial expression, and weak binding to Peptidyl-tRNA for PTRHD1. Additionally, we report binding to tRNA but absence of Peptidyl-tRNA hydrolase activity. Thus, PTRHD1 is not a Pth and the functional consequence of nucleotide binding remains undefined.
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A Highly Adaptable Method for Quantification of Peptidyl-tRNA Hydrolase Activity
Journal of analytical and bioanalytical techniques, 2015Co-Authors: W. Blake Holloway, Hana Mcfeeters, Adam M Powell, Gnana S Nidadavolu, Robert L. McfeetersAbstract:The emerging importance of Peptidyl-tRNA hydrolase (Pth) enzymes necessitates the need for a widely applicable functional assay to further studies of this important enzyme family. Previously reported methods for monitoring Pth function suffer from limitations of cost, time, substrate availability, and application compatibility. Herein we present a new method for the rapid and precise characterization of Pth activity. The method is applicable for use with specific or bulk Peptidyl-tRNA, any Pth enzyme, and a range of reaction conditions including solvent additives. The method also allows for semi-automated quantitative assessment of Peptidyl-tRNA cleavage. No specialized equipment, harmful reagents, or time-consuming techniques are required. We use the new method to characterize Pth activity, determine enzyme kinetic parameters, screen for inhibitors, and determine inhibitory parameters.
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Current Methods for Analysis of Enzymatic Peptidyl-tRNA Hydrolysis
Journal of analytical and bioanalytical techniques, 2014Co-Authors: Hana Mcfeeters, Robert L. McfeetersAbstract:Understanding Peptidyl-tRNA and the enzymes responsible for recycling them has come from the ability to detect and quantify enzymatic Peptidyl-tRNA hydrolysis. The methods available to study removal of peptides from tRNA have evolved considerably. Radioactive [14C] amino acids were first implemented to monitor cleavage of the peptide-nucleotide ester bond of uniform Peptidyl-tRNA substrates. Later, Northern blots with radiolabeled oligonucleotide probes were used to observe cleavage of specific Peptidyl-tRNAs or individual tRNA from bulk Peptidyl-tRNA populations. Finally, the use of fluorescently labeled amino acids was introduced, which could be coupled to anisotropy or PAGE readouts. Here we review the methods for quantification and analysis of enzymatic Peptidyl-tRNA hydrolysis and summarize their inherent advantages and disadvantages.
Sylvain Blanquet - One of the best experts on this subject based on the ideXlab platform.
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RNA-binding Site of Escherichia coli Peptidyl-tRNA Hydrolase
Journal of Biological Chemistry, 2011Co-Authors: Laurent Giorgi, M Fromant, Sylvain Blanquet, François Bontems, Caroline Aubard, Pierre PlateauAbstract:In a cell, Peptidyl-tRNA molecules that have prematurely dissociated from ribosomes need to be recycled. This work is achieved by an enzyme called Peptidyl-tRNA hydrolase. To characterize the RNA-binding site of Escherichia coli Peptidyl-tRNA hydrolase, minimalist substrates inspired from tRNAHis have been designed and produced. Two minisubstrates consist of an N-blocked histidylated RNA minihelix or a small RNA duplex mimicking the acceptor and TψC stem regions of tRNAHis. Catalytic efficiency of the hydrolase toward these two substrates is reduced by factors of 2 and 6, respectively, if compared with N-acetyl-histidyl-tRNAHis. In contrast, with an N-blocked histidylated microhelix or a tetraloop missing the TψC arm, efficiency of the hydrolase is reduced 20-fold. NMR mapping of complex formation between the hydrolase and the small RNA duplex indicates amino acid residues sensitive to RNA binding in the following: (i) the enzyme active site region; (ii) the helix-loop covering the active site; (iii) the region including Leu-95 and the bordering residues 111–117, supposed to form the boundary between the tRNA core and the peptidyl-CCA moiety-binding sites; (iv) the region including Lys-105 and Arg-133, two residues that are considered able to clamp the 5′-phosphate of tRNA, and (v) the positively charged C-terminal helix (residues 180–193). Functional value of these interactions is assessed taking into account the catalytic properties of various engineered protein variants, including one in which the C-terminal helix was simply subtracted. A strong role of Lys-182 in helix binding to the substrate is indicated.
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Peptidyl-tRNA hydrolase from Sulfolobus solfataricus
Nucleic Acids Research, 2003Co-Authors: M Fromant, Pierre Plateau, Maria-laura Ferri-fioni, Sylvain BlanquetAbstract:An enzyme capable of liberating functional tRNALys from Escherichia coli diacetyl-lysyl-tRNALys was purified from the archae Sulfolobus solfataricus. Contrasting with the specificity of peptidyl- tRNA hydrolase (PTH) from E.coli, the S.solfataricus enzyme readily accepts E.coli formyl-methionyl-tRNAfMet as a substrate. N-terminal sequencing of this enzyme identifies a gene that has homologs in the whole archaeal kingdom. Involvement of this gene (SS00175) in the recycling of Peptidyl-tRNA is supported by its capacity to complement an E.coli strain lacking PTH activity. The archaeal gene, the product of which appears markedly different from bacterial PTHs, also has homologs in all the available eukaryal genomes. Since most of the eukaryotes already display a bacterial-like PTH gene, this observation suggests the occurrence in many eukaryotes of two distinct PTH activities, either of a bacterial or of an archaeal type. Indeed, the bacterial- and archaeal-like genes encoding the two full-length PTHs of Saccharomyces cerevisiae, YHR189w and YBL057c, respectively, can each rescue the growth of an E.coli strain lacking endogeneous PTH. In vitro assays confirm that the two enzymes ensure the recycling of tRNALys from diacetyl-lysyl-tRNALys. Finally, the growth of yeast cells in which either YHR189w or YBL057c has been disrupted was compared under various culture conditions. Evidence is presented that YHR189w, the gene encoding a bacterial-like PTH, should be involved in mitochondrial function.
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Crystal structure at 1.2 Å resolution and active site mapping of Escherichia coli Peptidyl-tRNA hydrolase
The EMBO Journal, 1997Co-Authors: Emmanuelle Schmitt, Yves Mechulam, M Fromant, Pierre Plateau, Sylvain BlanquetAbstract:Peptidyl‐tRNA hydrolase activity from Escherichia coli ensures the recycling of peptidyl‐tRNAs produced through abortion of translation. This activity, which is essential for cell viability, is carried out by a monomeric protein of 193 residues. The structure of crystalline peptidyl‐tRNA hydrolase could be solved at 1.2 A resolution. It indicates a single α/β globular domain built around a twisted mixed β‐sheet, similar to the central core of an aminopeptidase from Aeromonas proteolytica . This similarity allowed the characterization by site‐directed mutagenesis of several residues of the active site of peptidyl‐tRNA hydrolase. These residues, strictly conserved among the known peptidyl‐tRNA hydrolase sequences, delineate a channel which, in the crystal, is occupied by the C‐end of a neighbouring peptidyl‐tRNA hydrolase molecule. Hence, several main chain atoms of three residues belonging to one peptidyl‐tRNA hydrolase polypeptide establish contacts inside the active site of another peptidyl‐tRNA hydrolase molecule. Such an interaction is assumed to represent the formation of a complex between the enzyme and one product of the catalysed reaction.
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Role of the 1-72 base pair in tRNAs for the activity of Escherichia coli Peptidyl-tRNA hydrolase
Nucleic Acids Research, 1993Co-Authors: Sophie Dutka, Yves Mechulam, Thierry Meinnel, Christine Lazennec, Sylvain BlanquetAbstract:Abstract Previous work by Schulman and Pelka (1975) J. Biol. Chem. 250, 542-547, indicated that the absence of a pairing between the bases 1 and 72 in initiator tRNA(fMet) explained the relatively small activity of Peptidyl-tRNA hydrolase towards N-acetyl-methionyl-tRNA(fMet). In the present study, the structural requirements for the sensitivity of an N-acetyl-aminoacyl-tRNA to Escherichia coli Peptidyl-tRNA hydrolase activity have been further investigated. Ten derivatives of tRNA(fMet) with various combinations of bases at positions 1 and 72 in the acceptor stem have been produced, aminoacylated and chemically acetylated. The release of the aminoacyl moiety from these tRNA derivatives was assayed in the presence of Peptidyl-tRNA hydrolase purified from an overproducing strain. tRNA(fMet) derivatives with either C1A72, C1C72, U1G72, U1C72 or A1C72 behaved as poor substrates of the enzyme, as compared to those with C1G72, U1A72, G1C72, A1U72 or G1U72. With the exception of U1G72, it could be therefore concluded that the relative resistance of tRNA(fMet) to Peptidyl-tRNA hydrolase did not depend on a particular combination of nucleotides at positions 1 and 72, but rather reflected the absence of a base pairing at these positions. In a second series of experiments, the unpairing of the 1 and 72 bases, created with C-A or A-C bases, instead of G-C in methionyl-tRNA(mMet) or in valyl-tRNA(Val1), was shown to markedly decrease the rate of hydrolysis catalysed by Peptidyl-tRNA hydrolase. Altogether, the data indicate that the stability of the 1-72 pair governs the degree of sensitivity of a Peptidyl-tRNA to Peptidyl-tRNA hydrolase.
Gabriel Guarneros - One of the best experts on this subject based on the ideXlab platform.
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Protein synthesis factors (RF1, RF2, RF3, RRF, and tmRNA) and Peptidyl-tRNA hydrolase rescue stalled ribosomes at sense codons.
Journal of Molecular Biology, 2012Co-Authors: Serafín Vivanco-domínguez, José G. Bueno-martínez, Gloria León-Ávila, Nobuhiro Iwakura, Akira Kaji, Hideko Kaji, Gabriel GuarnerosAbstract:Abstract During translation, ribosomes stall on mRNA when the aminoacyl-tRNA to be read is not readily available. The stalled ribosomes are deleterious to the cell and should be rescued to maintain its viability. To investigate the contribution of some of the cellular translation factors on ribosome rescuing, we provoked stalling at AGA codons in mutants that affected the factors and then analyzed the accumulation of oligopeptidyl (peptides of up to 6 amino acid residues, oligopep-)-tRNA or polypeptidyl (peptides of more than 300 amino acids in length, polypep-)-tRNA associated with ribosomes. Stalling was achieved by starvation for aminoacyl-tRNA Arg4 upon induced expression of engineered lac Z (β-ga lac tosidase) reporter gene harboring contiguous AGA codons close to the initiation codon or at internal codon positions together with minigene ATGAGATAA accompanied by reduced Peptidyl-tRNA hydrolase (Pth). Our results showed accumulations of Peptidyl-tRNA associated with ribosomes in mutants for release factors (RF1, RF2, and RF3), ribosome recycling factor (RRF), Pth, and transfer-messenger RNA (tmRNA), implying that each of these factors cooperate in rescuing stalled ribosomes. The role of these factors in ribosome releasing from the stalled complex may vary depending on the length of the peptide in the Peptidyl-tRNA. RF3 and RRF rescue stalled ribosomes by “drop-off” of Peptidyl-tRNA, while RF1, RF2 (in the absence of termination codon), or Pth may rescue by hydrolyzing the associated Peptidyl-tRNA. This is followed by the disassembly of the ribosomal complex of tRNA and mRNA by RRF and elongation factor G.
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Excess of charged tRNALys maintains low levels of Peptidyl-tRNA hydrolase in pth(Ts) mutants at a non-permissive temperature
Nucleic Acids Research, 2006Co-Authors: Serafín Vivanco-domínguez, Luis R. Cruz-vera, Gabriel GuarnerosAbstract:Cellular changes have been monitored during the suppression, mediated by the overproduction of tRNA Lys , of thermosensitivity in Escherichia coli strain AA7852 carrying a mutation in Peptidyl-tRNA hydrolase (Pth) encoded by the pth(Ts) gene. The presence in AA7852 cells of a plasmid bearing lysV gene helped to maintain low levels of the unstable Pth(Ts) protein and to preserve the viability of the mutant line at 41 � C whereas plasmids bearing other tRNA genes were ineffective. At 32 � C the excess of tRNA Lys did not alter the percentages of the free-, charged- or Peptidyl-tRNA Lys species compared with those found in strains that did not overproduce tRNA Lys .A t 41 � C, however, despite increases in the level of Peptidyl-tRNA Lys , the excess tRNA Lys helped to maintain the concentration of charged-tRNA Lys at a level comparable with that found in non-overproducer cells grown at a permissive temperature. In addition, the excess tRNA Lys at 41 � C provoked a reduction in the concentrations of various Peptidyl-tRNAs, which normally accumulate in pth(Ts) cells, and a proportional increase in the concentrations of the corresponding aminoacyl-tRNAs. The possible mechanism of rescue due to the overexpression of tRNA Lys and the causes of tRNA Lys starvation in
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Ribosome stalling and Peptidyl-tRNA drop-off during translational delay at AGA codons
Nucleic Acids Research, 2004Co-Authors: Luis R. Cruz-vera, Marco Antonio Magos-castro, Efraín Zamora-romo, Gabriel GuarnerosAbstract:Minigenes encoding the peptide Met–Arg–Arg have been used to study the mechanism of toxicity of AGA codons proximal to the start codon or prior to the termination codon in bacteria. The codon sequences of the ‘mini-ORFs’ employed were initiator, combinations of AGA and CGA, and terminator. Both, AGA and CGA are low-usage Arg codons in ORFs of Escherichia coli but, whilst AGA is translated by the scarce tRNAArg4, CGA is recognized by the abundant tRNAArg2. Overexpression of minigenes harbouring AGA in the third position, next to a termination codon, was deleterious to the cell and led to the accumulation of Peptidyl-tRNAArg4 and of the Peptidyl-tRNA cognate to the preceding CGA or AGA Arg triplet. The minigenes carrying CGA in the third position were not toxic. Minigene-mediated toxicity and Peptidyl-tRNA accumulation were suppressed by overproduction of tRNAArg4 but not by overproduction of Peptidyl-tRNA hydrolase, an enzyme that is only active on substrates that have been released from the ribosome. Consistent with these findings, Peptidyl-tRNAArg4 was identified to be mainly associated with ribosomes in a stand-by complex. These and previous results support the hypothesis that the primary mechanism of inhibition of protein synthesis by AGA triplets in pth+ cells involves sequestration of tRNAs as Peptidyl-tRNA on the stalled ribosome.
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The Rate of Peptidyl-tRNA Dissociation from the Ribosome during Minigene Expression Depends on the Nature of the Last Decoding Interaction
Journal of Biological Chemistry, 2003Co-Authors: L. Rogelio Cruz-vera, Elena Hernández-ramón, Bernardo Perez-zamorano, Gabriel GuarnerosAbstract:Abstract The expression of some very short open reading frames (ORFs) in Escherichia coli results in Peptidyl-tRNA accumulation that is lethal to cells defective in Peptidyl-tRNA hydrolase activity. In an attempt to understand the factors that affect this phenotype, we have surveyed the toxicity of a complete set of two-codon ORFs cloned as minigenes in inducible expression vectors. The minigenes were tested in hydrolase-defective hosts and classified according to their degree of toxicity. In general, minigenes harboring codons belonging to the same box in the standard table of the genetic code mediated similar degrees of toxicity. Moreover, the levels of Peptidyl-tRNA accumulation for synonymous minigenes decoded by the same tRNA were comparable. However, two exceptions were observed: (i) expression of minigenes harboring the Arg codons CGA, CGU, and CGC, resulted in the accumulation of different levels of the unique Peptidyl-tRNAArg-2 and (ii) the toxicity of minigenes containing CUG and UCU codons, each recognized by two different tRNAs, depended on Peptidyl-tRNA accumulation of only one of them. Non-toxic, or partly toxic, minigenes prompted higher accumulation levels of Peptidyl-tRNA upon deprivation of active RF1, implying that translation termination occurred efficiently. Our data indicate that the nature of the last decoding tRNA is crucial in the rate of Peptidyl-tRNA release from the ribosome.
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Orthologs of a novel archaeal and of the bacterial peptidyl–tRNA hydrolase are nonessential in yeast
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Guillermina Rosas-sandoval, Gabriel Guarneros, L. Rogelio Cruz-vera, Alexandre Ambrogelly, Jesse Rinehart, David E. Graham, Karl O. Stetter, Dieter SöllAbstract: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-[14C]lysyl–tRNA and acetyl-[14C]phenylalanyl–tRNA. On transformation of an Escherichia coli pthts 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.
Reynald Gillet - One of the best experts on this subject based on the ideXlab platform.
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The task force that rescues stalled ribosomes in bacteria.
Trends in Biochemical Sciences, 2017Co-Authors: Emmanuel Giudice, Reynald GilletAbstract:In bacteria, the main quality control mechanism for rescuing ribosomes that have arrested during translation is trans-translation, performed by transfer-mRNA (tmRNA) associated with small protein B (SmpB). Intriguingly, this very elegant mechanism is not always necessary to maintain cell viability, suggesting the existence of alternatives. Other rescue systems have recently been discovered, revealing a far more complicated story than expected. These include the alternative ribosome rescue factors ArfA and ArfB, the elongation factors EF4 and EF-P, the Peptidyl-tRNA hydrolase Pth, and several protein synthesis factors. These discoveries make it possible to describe a large network of factors dedicated to ribosome rescue, thus ensuring cell survival during stresses that induce ribosome stalling.