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Peptidyl-tRNA hydrolase is involved in lambda inhibition of host protein synthesis.

ABSTRACT: Escherichia coli rap mutants do not support vegetative growth of bacteriophage lambda and die upon transcription of lambda DNA bar sites. Bacteria harbouring a pth(ts) mutation synthesize thermosensitive peptidyl-tRNA hydrolase (Pth) and die at 42 degrees C from a defect in protein synthesis. We present evidence that both rap and pth(ts) mutations affect the same gene: (i) peptidyl-tRNA hydrolase activity was found to be defective in rap mutants; (ii) at a threshold temperature, pth cells, like rap mutants, prevented lambda growth and were killed by transcription of cloned bar sites; (iii) sequencing a 1600 bp DNA fragment comprising both loci revealed an ORF located within the limits set by a complementation analysis and encoding a putative polypeptide of 21 kDa; (iv) cloning and sequencing of rap and pth(ts) mutant DNAs both revealed single nucleotide transitions from the wild type ORF sequence, resulting in Arg134 to His and Gly101 to Asp changes respectively. Analysis of plasmid-directed proteins identified a polypeptide of approximately 21 kDa; the N-terminal sequence, amino acid composition and isoelectric point of this protein match those expected from the ORF nucleotide sequence. We propose that Pth activity, directly or indirectly, is the target for lambda bar RNA leading to rap cell death.

SUBMITTER: Garcia-Villegas MR

PROVIDER: S-EPMC453084 | biostudies-other | 1991 Nov

REPOSITORIES: biostudies-other

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Peptidyl-tRNA hydrolase is involved in lambda inhibition of host protein synthesis.

García-Villegas M R MR De La Vega F M FM Galindo J M JM Segura M M Buckingham R H RH Guarneros G G

The EMBO journal 19911101 11

Escherichia coli rap mutants do not support vegetative growth of bacteriophage lambda and die upon transcription of lambda DNA bar sites. Bacteria harbouring a pth(ts) mutation synthesize thermosensitive peptidyl-tRNA hydrolase (Pth) and die at 42 degrees C from a defect in protein synthesis. We present evidence that both rap and pth(ts) mutations affect the same gene: (i) peptidyl-tRNA hydrolase activity was found to be defective in rap mutants; (ii) at a threshold temperature, pth cells, like ...[more]

PMID: 1833189

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Project description:Protein biosynthesis is a central process in all living cells that is catalyzed by a complex molecular machine─the ribosome. This process is termed translation because the language of nucleotides in mRNAs is translated into the language of amino acids in proteins. Transfer RNA (tRNA) molecules charged with amino acids serve as adaptors and recognize codons of mRNA in the decoding center while simultaneously the individual amino acids are assembled into a peptide chain in the peptidyl transferase center (PTC). As the nascent peptide emerges from the ribosome, it is threaded through a long tunnel referred to as a nascent peptide exit tunnel (NPET). The PTC and NPET are the sites targeted by many antibiotics and are thus of tremendous importance from a biomedical perspective and for drug development in the pharmaceutical industry.Researchers have achieved much progress in characterizing ribosomal translation at the molecular level; an impressive number of high-resolution structures of different functional and inhibited states of the ribosome are now available. These structures have significantly contributed to our understanding of how the ribosome interacts with its key substrates, namely, mRNA, tRNAs, and translation factors. In contrast, much less is known about the mechanisms of how small molecules, especially antibiotics, affect ribosomal protein synthesis. This mainly concerns the structural basis of small molecule-NPET interference with cotranslational protein folding and the regulation of protein synthesis. Growing biochemical evidence suggests that NPET plays an active role in the regulation of protein synthesis.Much-needed progress in this field is hampered by the fact that during the preparation of ribosome complexes for structural studies (i.e., X-ray crystallography, cryoelectron microscopy, and NMR spectroscopy) the aminoacyl- or peptidyl-tRNAs are unstable and become hydrolyzed. A solution to this problem is the application of hydrolysis-resistant mimics of aminoacyl- or peptidyl-tRNAs.In this Account, we present an overview of synthetic methods for the generation of peptidyl-tRNA analogs. Modular approaches have been developed that combine (i) RNA and peptide solid-phase synthesis on 3'-aminoacylamino-adenosine resins, (ii) native chemical ligations and Staudinger ligations, (iii) tailoring of tRNAs by the selective cleavage of natural native tRNAs with DNAzymes followed by reassembly with enzymatic ligation to synthetic peptidyl-RNA fragments, and (iv) enzymatic tailing and cysteine charging of the tRNA to obtain modified CCA termini of a tRNA that are chemically ligated to the peptide moiety of interest. With this arsenal of tools, in principle, any desired sequence of a stably linked peptidyl-tRNA mimic is accessible. To underline the significance of the synthetic conjugates, we briefly point to the most critical applications that have shed new light on the molecular mechanisms underlying the context-specific activity of ribosome-targeting antibiotics, ribosome-dependent incorporation of multiple consecutive proline residues, the incorporation of d-amino acids, and tRNA mischarging.Furthermore, we discuss new types of stably charged tRNA analogs, relying on triazole- and squarate (instead of amide)-linked conjugates. Those have pushed forward our mechanistic understanding of nonribosomal peptide synthesis, where aminoacyl-tRNA-dependent enzymes are critically involved in various cellular processes in primary and secondary metabolism and in bacterial cell wall synthesis.

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Peptidyl-tRNA hydrolase is involved in lambda inhibition of host protein synthesis.

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Peptidyl-tRNA hydrolase is involved in lambda inhibition of host protein synthesis.

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