Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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The gene, prs, encoding phosphoribosylpyrophosphate (PRPP) synthetase of Escherichia coli was isolated from a library of E. coli genes cloned in the bacteriophage lambda D69 vector. A strain with a temperature-lethal defect in PRPP synthetase, (prs-2), was used as the host and cloning was performed by lysogenic complementation. The prs gene resided on a 5.6 kilobase-pair (kbp) DNA fragment generated by hydrolysis with restriction endonuclease BamHI. The nearby gene pth, encoding peptidyl-tRNA hydrolase, was also on this fragment. Subcloning of the fragment in the multi-copy plasmid pBR322 and subsequent deletion of parts of the insert resulted in a 1.7 kbp DNA fragment containing the entire prs gene. Bacterial strains harbouring prs-bearing plasmids showed up to 50-fold increased PRPP synthetase activity. The PRPP synthetase subunit was identified by analysis of plasmid-harbouring minicells and the subunit molecular mass established as 33,000 daltons. Analysis, by the minicell procedure, of plasmids with deletions extending into the prs gene established the direction of transcription as counterclockwise. A putative leader sequence of approximately 400 bp preceded the coding sequence. By deletion analysis and by cloning fragments of this leader sequence in a galK expression vector it was found to contain the prs promoter as well as a potential transcription termination site.
Mol Gen Genet 1985
PMID:Cloning and characterization of the prs gene encoding phosphoribosylpyrophosphate synthetase of Escherichia coli. 300 29

Peptidyl-tRNA may dissociate preferentially from ribosomes during protein synthesis when it is inappropriate to, does not correctly complement, the messenger RNA. To test this idea, growing cultures of Escherichia coli were treated with streptomycin to increase the frequency of errors during protein synthesis. Since the treated cells had a temperature-sensitive peptidyl-tRNA hydrolase and could not destroy dissociated peptidyl-tRNA, it was possible to measure the rate of its accumulation after raising the temperature to non-permissive conditions. Both low and high doses of streptomycin enhanced the rate of dissociation and accumulation of peptidyl-tRNA. The rank order of rates of dissociation/accumulation of various isoaccepting tRNA families was not significantly altered by the drug treatment. We concluded that streptomycin stimulated a normal pathway for dissociation of peptidyl-tRNA. Two streptomycin- resistant strains of E. coli had higher rates of dissociation of peptidyl-tRNA than did their sensitive parent strain. When treated with high doses of the drug, the resistant strains showed slightly reduced rates of dissociation of peptidyl-tRNA. These results were interpreted in terms of a two state, two site model for protein synthesis: streptomycin enhances the binding of aminoacyl-tRNA to a tight state of the ribosome A site; the strA mutation enhances translocation to a loose state of the ribosome P site.
Mol Gen Genet 1984
PMID:Dissociation of peptidyl-tRNA from ribosomes is perturbed by streptomycin and by strA mutations. 620 86

Derivatives of isogenic stringent (relA+) and relaxed (relA) strains of Escherichia coli were compared in respect of rates of the dissociation of peptidyl-tRNA from ribosomes during protein synthesis. The derivatives both contained a mutant pth gene which rendered temperature-sensitive their peptidyl-tRNA hydrolase (E.C. 3.1.1.29) activities. After shifting from permissive 30 degrees C to non-permissive 40 degrees C, dissociated peptidyl-tRNA accumulated and was assayed chemically or by its cytotoxic effects. In unperturbed (except for the temperature shift) cultures the relA strain accumulated peptidyl-tRNA significantly more slowly than did its relA+ isogenic cousin. Both strains responded approximately equally to non-lethal doses of erythromycin or to starvation for amino acids. Both these perturbations enhanced the dissociation and accumulation of peptidyl-tRNA. While growing at 30 degrees C, both strains responded significantly to a nutritional downshift from growth in medium containing glucose plus amino acids to growth in medium containing only amino acids. Taken together the results suggested that different intracellular concentrations of ppGpp in unperturbed cells, attributable to the different relA alleles, could account for the differences in dissociation and accumulation of peptidyl-tRNA. Our observation of a lower rate of dissociation of peptidyl-tRNA in the relA strain, coupled with the reported lower intracellular ppGpp and lower accuracy of protein synthesis, is consistent with the idea that relA strains have less efficient ribosomal editing of erroneous peptidyl-tRNA.
Mol Gen Genet 1983
PMID:Tests of the ribosome editor hypothesis. II. Relaxed (relA) and stringent (relA+) E. coli differ in rates of dissociation of peptidyl-tRNA from ribosomes. 634 73

Escherichia coli mutants defective in peptidyl-tRNA hydrolase activity are unable to maintain bacteriophage lambda vegetative growth. Phage mutants, named bar, overcome the host limitation to support viral growth. Multicopy expression of lambda wild-type bar regions is deleterious to hydrolase-defective cells because it provokes arrest of protein synthesis. We noticed that the bar regions include minigenes whose transcripts would contain a Shine-Dalgarno-like sequence appropriately spaced for translation from a two codon open reading frame. To investigate the mechanism of bar inhibition, we asked if transcripts of the barI region function as mRNAs in their ribosomal interactions. We found that bar-containing RNA associates with ribosomes, forms ternary initiation complexes, yields a toeprint signal, and can be removed from ribosomes by run-off translation, as authentic mRNA. Since bar-containing RNA has the properties of a messenger, we propose that its translation leads to drop-off and accumulation of peptidyl-tRNA in pth-defective cells. Starvation of the tRNA(s) sequestered in pepidyl-tRNA(s) eventually causes inhibition of protein synthesis.
J Mol Biol 1997 Jun 06
PMID:Inhibition of Escherichia coli protein synthesis by abortive translation of phage lambda minigenes. 919 Oct 62

A novel function of initiation factors IF1 and IF2 in Escherichia coli translation has been identified. It is shown that these factors efficiently catalyse dissociation of peptidyl-tRNAs with polypeptides of different length from the P-site of E. coli ribosomes, and that the simultaneous presence of both factors is required for induction of drop-off. The factor-induced drop-off occurs with both sense and stop codons in the A-site and competes with peptide elongation or termination. The efficiency with which IF1 and IF2 catalyse drop-off decreases with increasing length of the nascent polypeptide, but is quite significant for hepta-peptidyl-tRNAs, the longest polypeptide chains studied. In the absence of IF1 and IF2 the rate of drop-off varies considerably for different peptidyl-tRNAs, and depends both on the length and sequence of the nascent peptide. Efficient factor-catalysed drop-off requires GTP but not GTP hydrolysis, as shown in experiments without guanine nucleotides, with GDP or with the non-cleavable analogue GMP-PNP.Simultaneous overexpression of IF1 and IF2 in vivo inhibits cell growth specifically in some peptidyl-tRNA hydrolase deficient mutants, suggesting that initiation factor-catalysed drop-off of peptidyl-tRNA can occur on a significant scale in the bacterial cell. Consequences for the bacterial physiology of this previously unknown function of IF1 and IF2 are discussed.
J Mol Biol 1998 Aug 14
PMID:Initiation factors IF1 and IF2 synergistically remove peptidyl-tRNAs with short polypeptides from the P-site of translating Escherichia coli ribosomes. 969 45

Mutants of Escherichia coli partially deficient in peptidyl-tRNA hydrolase are killed by the expression of certain very short open reading frames (mini-genes), encoded by the wild-type bar regions of phage lambda. According to the current hypothesis, protein synthesis is shut off, and the host cells die, after essential tRNA species become sequestered due to abnormal translation termination (drop-off) of mini-gene-encoded peptides as peptidyl-tRNA. Here we study variants of bar mini-genes, both in vivo and in vitro, in order to identify the structural elements that influence this inhibition of protein synthesis. Three parameters were measured during the expression of these variants: the rates of normal translation termination, peptidyl-tRNA dissociation from the ribosome and hydrolysis of peptidyl-tRNA by peptidyl-tRNA hydrolase were measured. Previous observations that RRF, EF-G and RF3 stimulated drop-off were confirmed and extended; stimulation by these factors can reach 30-fold. Both factor-stimulated and spontaneous drop-off depended on the nature of the stop signal. The degree of inhibition of cell growth following induction of mini-gene expression could be accounted for in terms of a toxicity index comprising the three parameters above. Inhibition was greatly reduced in cells lacking RF3. Mini-genes with more efficient Shine/Dalgarno sequences killed cells even with normal peptidyl-tRNA hydrolase activity. It is proposed that the retranslation by ribosomes of mini-gene transcripts with efficient ribosome binding (Shine/Dalgarno) sequences strongly contributes to the inhibitory effects of mini-gene expression on protein synthesis.
J Mol Biol 1999 Aug 27
PMID:Shutdown in protein synthesis due to the expression of mini-genes in bacteria. 1045 86

Bacteriophage lambda is unable to grow vegetatively on Escherichia coli mutants defective in peptidyl-tRNA hydrolase (Pth) activity. Mutations which allow phage growth on the defective host have been located at regions named bar in the lambda genome. Expression of wild-type bar regions from plasmid constructs results in inhibition of protein synthesis and lethality to Pth-defective cells. Two of these wild-type bar regions, barI+ and barII+, contain minigenes with similar AUG-AUA-stop codon sequences preceded by different Shine-Dalgarno (SD) and spacer regions. The induced expression of barI+ and barII+ regions from plasmid constructs resulted in similar patterns of protein synthesis inhibition and cell growth arrest. Therefore, these deleterious effects may stem from translation of the transcripts containing the minigene two-codon 'ORF' (open reading frame). To test for this possibility, we assayed the effect of point mutations within the barI minigene. The results showed that a base pair substitution within the SD and the two-codon 'ORF' sequences affected protein synthesis and cell growth inhibition. In addition, mRNA stability was altered in each mutant. Higher mRNA stability correlated with the more toxic minigenes. We argue that this effect may be caused by ribosome protection of the mRNA in paused complexes as a result of deficiency of specific tRNA.
Mol Microbiol 2001 Jan
PMID:Increased bar minigene mRNA stability during cell growth inhibition. 1113 57

To analyse the mechanism by which rare codons near the initiation codon inhibit cell growth and protein synthesis, we used the bacteriophage lambda int gene or early codon substitution derivatives. The lambda int gene has a high frequency of rare ATA, AGA and AGG codons; two of them (AGA AGG) located at positions 3 and 4 of the int open reading frame (ORF). Escherichia coli pth (rap) cells, which are defective in peptidyl-tRNA hydrolase (Pth) activity, are more susceptible to the inhibitory effects of int expression as compared with wild-type cells. Cell growth and Int protein synthesis were enhanced by overexpression of Pth and tRNAArg4 cognate to AGG and AGA but not of tRNAIle2a specific for ATA. The increase of Int protein synthesis also takes place when the rare arginine codons AGA and AGG at positions 3 and 4 are changed to common arginine CGT or lysine AAA codons but not to rare isoleucine ATA codons. In addition, overexpression of int in Pth defective cells provokes accumulation of peptidyl-tRNAArg4 in the soluble fraction. Therefore, cell growth and Int synthesis inhibition may be due to ribosome stalling and premature release of peptidyl-tRNAArg4 from the ribosome at the rare arginine codons of the first tandem, which leads to cell starvation for the specific tRNA.
Mol Microbiol 2003 Aug
PMID:The pair of arginine codons AGA AGG close to the initiation codon of the lambda int gene inhibits cell growth and protein synthesis by accumulating peptidyl-tRNAArg4. 1289 27

Chloroplast RNA splicing 2 (CRS2) is a nuclear-encoded protein required for the splicing of nine group II introns in maize chloroplasts. CRS2 functions in the context of splicing complexes that include one of two CRS2-associated factors (CAF1 and CAF2). The CRS2-CAF1 and CRS2-CAF2 complexes are required for the splicing of different subsets of CRS2-dependent introns, and they bind tightly and specifically to their genetically defined intron targets in vivo. The CRS2 amino acid sequence is closely related to those of bacterial peptidyl-tRNA hydrolases (PTHs). To identify the structural differences between CRS2 and bacterial PTHs responsible for CRS2's gains of CAF binding and intron splicing functions, we determined the structure of CRS2 by X-ray crystallography. The fold of CRS2 is the same as that of Escherichia coli PTH, but CRS2 has two surfaces that differ from the corresponding surfaces in PTH. One of these is more hydrophobic in CRS2 than in PTH. Site-directed mutagenesis of this surface blocked CRS2-CAF complex formation, indicating that it is the CAF binding site. The CRS2 surface corresponding to the putative tRNA binding face of PTH is considerably more basic than in PTH, suggesting that CRS2 interacts with group II intron substrates via this surface. Both the sequence and the structural context of the amino acid residues essential for peptidyl-tRNA hydrolase activity are conserved in CRS2, yet expression of CRS2 is incapable of rescuing a pth(ts)E.coli strain.
J Mol Biol 2005 Jan 07
PMID:Structural analysis of the group II intron splicing factor CRS2 yields insights into its protein and RNA interaction surfaces. 1556 10

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 Mycobacterium tuberculosis has been determined in three crystal forms. This structure and the structure of the enzyme from 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 polypeptide 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.
J Mol Biol 2007 Sep 07
PMID:Structural plasticity and enzyme action: crystal structures of mycobacterium tuberculosis peptidyl-tRNA hydrolase. 1761 20


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