Molecular Basis for the Temperature Sensitivity of
            <i>Escherichia coli pth</i>
            (Ts)

Cruz-Vera, Luis R.; Toledo, Ivonne; Hernández‐Sánchez, Javier; Guarneros, Gabriel

doi:10.1128/jb.182.6.1523-1528.2000

Cited by 37 publications

(47 citation statements)

References 31 publications

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“…The availability of the E. coli C600 pth ts mutant strain (2,17,26) provided the opportunity to test whether the archaeal pth2 gene would be active in E. coli and complement the temperature-sensitive phenotype. The E. coli C600 pth ts strain was transformed with plasmids carrying either the M. jannaschii pth2 (pGR518) or the E. coli pth gene (pGI01).…”

Section: Jannaschii Pth2 Complements the Temperature-sensitive Pthmentioning

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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Section: Jannaschii Pth2 Complements the Temperature-sensitive Pthmentioning

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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“…Bulk bacterial peptidyl-tRNAs were produced in C600 pth(Ts) cells [6]. A 3 mL LB starter was inoculated with C600 pth(Ts) cells and grown at 30°C.…”

Section: Preparation Of Peptidyl-trnamentioning

confidence: 99%

“…Furthermore, the method requires no specialized equipment or hazardous reagents. Of particular benefit, the method can employ heterogeneous peptidyltRNAs isolated from bacteria in high quantities with minimal resources [6,16]. We show the applicability of the method using two types of Pth enzymes, bacterial Pth1s and human Pth2.…”

Section: Introductionmentioning

confidence: 99%

“…In most bacteria, for example, there is a single highly conserved Pth1 enzyme that is solely responsible for recycling peptidyl-tRNA released from the ribosome. Loss of Pth1 function is lethal for these bacteria due to tRNA starvation [5,6]. Select bacterial species have an additional pth2 gene [7]; however functional redundancy has not been experimentally confirmed.…”

Section: Introductionmentioning

confidence: 99%

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A Highly Adaptable Method for Quantification of Peptidyl-tRNA Hydrolase Activity

Holloway¹,

McFeeters²,

Powell³

et al. 2015

J Anal Bioanal Tech

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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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“…Though distant from the active site proposed in the three-dimensional structure (Schmitt et al, 1997), this Gly residue is highly conserved in Pth from many different organisms. Recent work has shown that the mutation leads to a Pth protein that is unstable in vivo, at both permissive and non-permissive temperatures, but which shows a specific activity comparable to that of the wild-type enzyme (Cruz-Vera et al, 2000). The mutant enzyme seems not to be correctly folded and is subject to degradation by ClpP and Lon proteases (CruzVera et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

Suppression of thermosensitive peptidyl-tRNA hydrolase mutation in Escherichia coli by gene duplication

Menez

Rémy²,

Buckingham

2001

Microbiology

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Peptidyl-tRNA hydrolase (Pth) in Escherichia coli is required to recycle tRNA molecules that dissociate from the ribosome as peptidyl-tRNA during protein synthesis. At non-permissive temperatures, strains with a thermosensitive mutation affecting the enzyme accumulate peptidyl-tRNA, cease protein synthesis and die. The rate of reversion of this mutation to thermoresistance varies widely according to the genetic background of the cell and the temperature of selection ; under certain conditions, reversion can occur at rates approaching 10 N3 per cell per generation. In such revertants, a chromosomal pth gene can be replaced by an inactivated gene, restoring thermosensitive growth in most cases. PCR amplification experiments and Southern blots show the presence of both normal and inactivated copies of the gene, demonstrating that gene duplication has occurred in the revertants. Estimation of intracellular peptidyl-tRNA hydrolase by Western blotting confirms this explanation of the mechanism of high-frequency reversion to thermoresistance.

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Molecular Basis for the Temperature Sensitivity of Escherichia coli pth (Ts)

Cited by 37 publications

References 31 publications

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

A Highly Adaptable Method for Quantification of Peptidyl-tRNA Hydrolase Activity

Suppression of thermosensitive peptidyl-tRNA hydrolase mutation in Escherichia coli by gene duplication

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