Minigene-like inhibition of protein synthesis mediated by hungry codons near the start codon

Rare AGA or AGG codons close to the initiation codon inhibit protein synthesis by a tRNA-sequestering mechanism as toxic minigenes do. To further understand this mechanism, a parallel analysis of protein synthesis and peptidyl-tRNA accumulation was performed using both a set of lacZ constructs where AGAAGA codons were moved codon by codon from +2, +3 up to +7, +8 positions and a series of 3-8 codon minigenes containing AGAAGA codons before the stop codon. Beta-galactosidase synthesis from the AGAAGA lacZ constructs (in a Pth defective in vitro system without exogenous tRNA) diminished as the AGAAGA codons were closer to AUG codon. Likewise, beta-galactosidase expression from the reporter +7 AGA lacZ gene (plus tRNA, 0.25 microg/microl) waned as the AGAAGAUAA minigene shortened. Pth counteracted both the length-dependent minigene effect on the expression of beta-galactosidase from the +7 AGA lacZ reporter gene and the positional effect from the AGAAGA lacZ constructs. The +2, +3 AGAAGA lacZ construct and the shortest +2, +3 AGAAGAUAA minigene accumulated the highest percentage of peptidyl-tRNA(Arg4). These observations lead us to propose that hungry codons at early positions, albeit with less strength, inhibit protein synthesis by a minigene-like mechanism involving accumulation of peptidyl-tRNA.     

Evidence of bar minigene expression and tRNA2Ile sequestration as peptidyl-tRNA2Ile during lambda bacteriophage development

Lambda bacteriophage development is impaired in Escherichia coli cells defective for peptidyl (pep)-tRNA hydrolase (Pth). Single-base-pair mutations (bar(-)) that affect translatable two-codon open reading frames named bar minigenes (barI or barII) in the lambda phage genome promote the development of this phage in Pth-defective cells (rap cells). When the barI minigene is cloned and overexpressed from a plasmid, it inhibits protein synthesis and cell growth in rap cells by sequestering tRNA(2)(Ile) as pep-tRNA(2)(Ile). Either tRNA(2)(Ile) or Pth may reverse these effects. In this paper we present evidence that both barI and barII minigenes are translatable elements that sequester tRNA(2)(Ile) as pep-tRNA(2)(Ile). In addition, overexpression of the barI minigene impairs the development even of bar(-) phages in rap cells. Interestingly, tRNA or Pth may reestablish lambda phage development. These results suggest that lambda bar minigenes are expressed and tRNA(2)(Ile) is sequestered as pep-tRNA(2)(Ile) during lambda phage development.     

Inhibition of translation and cell growth by minigene expression

A random five-codon gene library was used to isolate minigenes whose expression causes cell growth arrest. Eight different deleterious minigenes were isolated, five of which had in-frame stop codons; the predicted expressed peptides ranged in size from two to five amino acids. Mutational analysis demonstrated that translation of the inhibitory minigenes is essential for growth arrest. Pulse-labeling experiments showed that expression of at least some of the selected minigenes results in inhibition of cellular protein synthesis. Expression of the deleterious minigenes in cells deficient in peptidyl-tRNA hydrolase causes accumulation of families of peptidyl-tRNAs corresponding to the last minigene codon; the inhibitory action of minigene expression could be suppressed by overexpression of the tRNA corresponding to the last sense codon in the minigene. Experimental data are compatible with the model that the deleterious effect of minigene expression is mediated by depletion of corresponding pools of free tRNAs.     

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

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.     

Protein synthesis factors (RF1, RF2, RF3, RRF, and tmRNA) and peptidyl-tRNA hydrolase rescue stalled ribosomes at sense codons

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 lacZ (β-galactosidase) 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.     

Ribosome bypassing elicited by tRNA depletion

Ribosome bypassing refers to the ability of the ribosome::peptidyl-tRNA complex to slide down the message without translation to a site several or dozens of nucleotides downstream and resume protein chain elongation there. The product is an isoform of a protein with a 'coding' gap corresponding to the region of the message which was bypassed. Previous work showed that ribosome bypassing was strongly stimulated at 'hungry' codons calling for a tRNA whose aminoacylation was limited. We have now used the 'minigene' phenomenon to ascertain whether depletion of the pool of specific isoacceptors has a similar effect. High level expression of plasmid-borne minigenes results in the sequestration as peptidyl-tRNA of tRNA cognate to the last triplet of the minigene, thereby limiting protein synthesis for lack of the tRNA in question. We find that induction of a minigene ending in AUA stimulates bypassing at an AUA codon, but not in a control sequence with AGA at the test position; induction of a minigene ending in AGA stimulates bypassing at the latter but not the former. Induction of the AUA minigene also stimulates both leftward and rightward frameshifting at 'shifty' sequences containing an AUA codon. The normal, background frequency of bypassing at an AUA codon is markedly reduced by increasing the cellular level of the tRNA which reads the codon. Thus, the frequency of bypassing can be increased or decreased by lowering or raising the concentration of a relevant tRNA isoacceptor. These observations suggest that the occurrence of ribosome bypassing reflects the length of the pause at a given codon.     

Ribosomal RNA and peptidyl-tRNA hydrolase: a peptide chain termination model for lambda bar RNA inhibition.

We propose here a model to explain the inhibition of bacteriophage lambda (lambda) vegetative growth and the killing of E coli cells defective in peptidyl-tRNA hydrolase (Pth) by lambda bar RNA. The model suggests that bar RNA, which contains a characteristic UGA triplet, base-pairs in an anti-parallel fashion with the 1199-1205 region of E coli 16S rRNA. In doing so, it prevents the required functioning of that region of 16S rRNA in UGA-specific peptide chain termination. Pth is implicated in peptide chain termination because a defect in Pth is required for the achievement of the bar RNA inhibitory effects. We make certain predictions that flow from the model, predictions involving suppression of nonsense mutations, and present preliminary experimental results that demonstrate the fulfillment of those predictions.     

Molecular basis for the temperature sensitivity of Escherichia coli pth(Ts)

The gene pth, encoding peptidyl-tRNA hydrolase (Pth), is essential for protein synthesis and viability of Escherichia coli. Two pth mutants have been studied in depth: a pth(Ts) mutant isolated as temperature sensitive and a pth(rap) mutant selected as nonpermissive for bacteriophage lambda vegetative growth. Here we show that each mutant protein is defective in a different way. The Pth(Ts) protein was very unstable in vivo, both at 43 degrees C and at permissive temperatures, but its specific activity was comparable to that of the wild-type enzyme, Pth(wt). Conversely, the mutant Pth(rap) protein had the same stability as Pth(wt), but its specific activity was low. The thermosensitivity of the pth(Ts) mutant, presumably, ensues after Pth(Ts) protein levels are reduced at 43 degrees C. Conditions that increased the cellular Pth(Ts) concentration, a rise in gene copy number or diminished protein degradation, allowed cell growth at a nonpermissive temperature. Antibiotic-mediated inhibition of mRNA and protein synthesis, but not of peptidyl-tRNA drop-off, reduced pth(Ts) cell viability even at a permissive temperature. Based on these results, we suggest that Pth(Ts) protein, being unstable in vivo, supports cell viability only if its concentration is maintained above a threshold that allows general protein synthesis.     

A physiological connection between tmRNA and peptidyl-tRNA hydrolase functions in Escherichia coli

The bacterial ssrA gene codes for a dual function RNA, tmRNA, which possesses tRNA-like and mRNA-like regions. The tmRNA appends an oligopeptide tag to the polypeptide on the P-site tRNA by a trans-translation process that rescues ribosomes stalled on the mRNAs and targets the aberrant protein for degradation. In cells, processing of the stalled ribosomes is also pioneered by drop-off of peptidyl-tRNAs. The ester bond linking the peptide to tRNA is hydrolyzed by peptidyl-tRNA hydrolase (Pth), an essential enzyme, which releases the tRNA and the aberrant peptide. As the trans-translation mechanism utilizes the peptidyl-transferase activity of the stalled ribosomes to free the tRNA (as opposed to peptidyl-tRNA drop-off), the need for Pth to recycle such tRNAs is bypassed. Thus, we hypothesized that tmRNA may rescue a defect in Pth. Here, we show that overexpression of tmRNA rescues the temperature-sensitive phenotype of Escherichia coli (pth^ts). Conversely, a null mutation in ssrA enhances the temperature-sensitive phenotype of the pth^ts strain. Consistent with our hypothesis, overexpression of tmRNA results in decreased accumulation of peptidyl-tRNA in E.coli. Furthermore, overproduction of tmRNA in E.coli strains deficient in ribosome recycling factor and/or lacking the release factor 3 enhances the rescue of pth^ts strains. We discuss the physiological relevance of these observations to highlight a major role of tmRNA in decreasing cellular peptidyl-tRNA load.     

Essential role of histidine 20 in the catalytic mechanism of Escherichia coli peptidyl-tRNA hydrolase

The peptidyl-tRNA hydrolase (Pth) enzyme plays an essential role in recycling tRNA from peptidyl-tRNA that has prematurely dissociated from the ribosome. In this study of Escherichia coli Pth, the critical role of histidine 20 was investigated by site-directed mutagenesis, stopped-flow kinetic measurements, and chemical modification. The histidine residue at position 20 is known to play an important role in the hydrolysis reaction, but stopped-flow fluorescence measurements showed that, although the His20Asn Pth mutant enzyme was unable to hydrolyze the substrate, the enzyme retained the ability to bind peptidyl-tRNA. Chemical modification of Pth with diethyl pyrocarbonate (DEPC) showed that a residue, with a pK(a) value of 6.3, was essential for substrate hydrolysis and that the stoichiometry of inhibition was 0.70 +/- 0.06 mol of DEPC/mol of enzyme, indicating that modification of only a single residue by DEPC was responsible for the loss of activity. Parallel chemical modification studies with the His20Asn and Asp93Asn mutant enzymes showed that this essential residue was His20. These studies indicate that histidine 20 acts as the catalytic base in the hydrolysis of peptidyl-tRNA by Pth.     

Codon-specific and general inhibition of protein synthesis by the tRNA-sequestering minigenes

The expression of minigenes in bacteria inhibits protein synthesis and cell growth. Presumably, the translating ribosomes, harboring the peptides as peptidyl-tRNAs, pause at the last sense codon of the minigene directed mRNAs. Eventually, the peptidyl-tRNAs drop off and, under limiting activity of peptidyl-tRNA hydrolase, accumulate in the cells reducing the concentration of specific aminoacylable tRNA. Therefore, the extent of inhibition is associated with the rate of starvation for a specific tRNA. Here, we used minigenes harboring various last sense codons that sequester specific tRNAs with different efficiency, to inhibit the translation of reporter genes containing, or not, these codons. A prompt inhibition of the protein synthesis directed by genes containing the codons starved for their cognate tRNA (hungry codons) was observed. However, a non-specific in vitro inhibition of protein synthesis, irrespective of the codon composition of the gene, was also evident. The degree of inhibition correlated directly with the number of hungry codons in the gene. Furthermore, a tRNA(Arg4)-sequestering minigene promoted the production of an incomplete beta-galactosidase polypeptide interrupted, during bacterial polypeptide chain elongation at sites where AGA codons were inserted in the lacZ gene suggesting ribosome pausing at the hungry codons.     

Yeast Pth2 is a UBL domain-binding protein that participates in the ubiquitin-proteasome pathway

Ubiquitin-like (UBL)-ubiquitin-associated (UBA) proteins such as Rad23 and Dsk2 mediate the delivery of polyubiquitinated proteins to the proteasome in the ubiquitin-proteasome pathway. We show here that budding yeast peptidyl-tRNA hydrolase 2 (Pth2), which was previously recognized as a peptidyl-tRNA hydrolase, is a UBL domain-binding protein that participates in the ubiquitin-proteasome pathway. Pth2 bound to the UBL domain of both Rad23 and Dsk2. Pth2 also interacted with polyubiquitinated proteins through the UBA domains of Rad23 and Dsk2. Pth2 overexpression caused an accumulation of polyubiquitinated proteins and inhibited the growth of yeast. Ubiquitin-dependent degradation was accelerated in the pth2Delta mutant and was retarded by overexpression of Pth2. Pth2 inhibited the interaction of Rad23 and Dsk2 with the polyubiquitin receptors Rpn1 and Rpn10 on the proteasome. Furthermore, Pth2 function involving UBL-UBA proteins was independent of its peptidyl-tRNA hydrolase activity. These results suggest that Pth2 negatively regulates the UBL-UBA protein-mediated shuttling pathway in the ubiquitin-proteasome system.     

Expression, purification, and characterization of peptidyl-tRNA hydrolase from Staphylococcus aureus.

Bacterial peptidyl-tRNA hydrolase (Pth) activity ensures the rapid recycling of peptidyl-tRNAs that result from premature termination of translation. Pth has been shown to be essential for growth in Escherichia coli suggesting that its homologue in Staphylococcus aureus is a potential molecular therapeutic target for the development of antibacterial agents. In this report we describe the cloning of a DNA fragment (573 bp) containing the pth gene from a S. aureus (strain ISP3) genomic DNA library. Analysis of the predicted polypeptide sequence from the pth gene showed that the protein shared complete conservation of the three residues thought to be involved in the active site of E. coli Pth. The gene was cloned into a pQE-60 expression vector and expressed in E. coli, and the resulting His-tagged Pth protein was purified to greater than 95% purity from the soluble portion of the E. coli lysate in a single chromatographic step. His-tagged Pth was shown to be biologically active by its ability to hydrolyze diacetyl-[(3)H]Lys-tRNA(Lys) in a time- and concentration-dependent manner. Optimum hydrolyzing activity of Pth occurred at a pH value of 7.0 and a MgCl(2) concentration of 5 mM. The K(m) of the diacetyl-[(3)H]-Lys-tRNA(Lys) substrate for S. aureus Pth was determined to be 2.8 microM. A far UV circular dichroism spectrum revealed that His-tagged S. aureus Pth appears to have a structured core predominated by beta-sheet.     

Abortive translation caused by peptidyl-tRNA drop-off at NGG codons in the early coding region of mRNA

In Escherichia coli the codons CGG, AGG, UGG or GGG (NGG codons) but not GGN or GNG (where N is non-G) are associated with low expression of a reporter gene, if located at positions +2 to +5. Induction of a lacZ reporter gene with any one of the NGG codons at position +2 to +5 does not influence growth of a normal strain, but growth of a strain with a defective peptidyl-tRNA hydrolase (Pth) enzyme is inhibited. The same codons, if placed at position +7, did not give this effect. Other codons, such as CGU and AGA, at location +2 to +5, did not give any growth inhibition of either the wild-type or the mutant strain. The inhibitory effect on the pth mutant strain by NGG codons at location +5 was suppressed by overexpression of the Pth enzyme from a plasmid. However, the overexpression of cognate tRNAs for AGG or GGG did not rescue from the growth inhibition associated with these codons early in the induced model gene. The data suggest that the NGG codons trigger peptidyl-tRNA drop-off if located at early coding positions in mRNA, thereby strongly reducing gene expression. This does not happen if these codons are located further down in the mRNA at position +7, or later.     

Structural basis for the substrate recognition and catalysis of peptidyl-tRNA hydrolase

Peptidyl-tRNA hydrolase (Pth) cleaves the ester bond between the peptide and the tRNA of peptidyl-tRNA molecules, which are produced by aborted translation, to recycle tRNA for further rounds of protein synthesis. Pth is ubiquitous in nature, and its enzymatic activity is essential for bacterial viability. We have determined the crystal structure of Escherichia coli Pth in complex with the tRNA CCA-acceptor-TΨC domain, the enzyme-binding region of the tRNA moiety of the substrate, at 2.4 Å resolution. In combination with site-directed mutagenesis studies, the structure identified the amino acid residues involved in tRNA recognition. The structure also revealed that Pth interacts with the tRNA moiety through the backbone phosphates and riboses, and no base-specific interactions were observed, except for the interaction with the highly conserved base G53. This feature enables Pth to accept the diverse sequences of the elongator-tRNAs as substrate components. Furthermore, we propose an authentic Pth:peptidyl-tRNA complex model and a detailed mechanism for the hydrolysis reaction, based on the present crystal structure and the previous studies’ results.     

Structural and functional insights into peptidyl-tRNA hydrolase

Peptidyl-tRNA hydrolase is an essential enzyme which acts as one of the rescue factors of the stalled ribosomes. It is an esterase that hydrolyzes the ester bond in the peptidyl-tRNA molecules, which are products of ribosome stalling. This enzyme is required for rapid clearing of the peptidyl-tRNAs, the accumulation of which in the cell leads to cell death. Over the recent years, it has been heralded as an attractive drug target for antimicrobial therapeutics. Two distinct classes of peptidyl-tRNA hydrolase, Pth and Pth2, have been identified in nature. This review gives an overview of the structural and functional aspects of Pth, along with its sequence and structural comparison among various species of bacteria. While the mode of binding of the substrate to Pth and the mechanism of hydrolysis are still speculated upon, the structure-based drug design using this protein as the target is still largely unexplored. This review focuses on the structural features of Pth, giving a direction to structure-based drug design on this protein.     

Crystal structure of a human peptidyl-tRNA hydrolase reveals a new fold and suggests basis for a bifunctional activity

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-A resolution as well as its catalytic properties. In contrast to the structure of Escherichia coli Pth, H. sapiens Pth2 has an alpha/beta fold with a four-stranded antiparallel beta-sheet in its core surrounded by two alpha-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.