Origins of minigene-dependent growth inhibition in bacterial cells

Heurgué-Hamard, Valérie; Dincbas, Vildan; Buckingham, Richard H.; Ehrenberg, Måns

doi:10.1093/emboj/19.11.2701

Cited by 51 publications

(67 citation statements)

References 32 publications

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“…Overexpression of the IF3 encoding gene, infC, is also predicted to be deleterious to the cell. It is known that ribosome recycling factor (RRF), and elongation factor G (EF-G) cooperate to release small peptidyl tRNAs from ribosomes stalled early in the translation of an open reading frame-this is particularly frequent during the first seven codons translated (Karimi et al 1998;Heurgue-Hamard et al 2000; for review, see Buchan and Stansfield 2007). This may help rapid abandonment of translation under conditions where aminoacyl tRNAs are in limiting supply, namely, as an amino acid starvation early warning system.…”

Section: Why Control By Negative Feedback: Translational Fidelity As mentioning

confidence: 99%

Autoregulatory systems controlling translation factor expression: Thermostat-like control of translational accuracy

Betney

Silva²,

Krishnan³

et al. 2010

RNA

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In both prokaryotes and eukaryotes, the expression of a large number of genes is controlled by negative feedback, in some cases operating at the level of translation of the mRNA transcript. Of particular interest are those cases where the proteins concerned have cell-wide function in recognizing a particular codon or RNA sequence. Examples include the bacterial translation termination release factor RF2, initiation factor IF3, and eukaryote poly(A) binding protein. The regulatory loops that control their synthesis establish a negative feedback control mechanism based upon that protein's RNA sequence recognition function in translation (for example, stop codon recognition) without compromising the accurate recognition of that codon, or sequence during general, cell-wide translation. Here, the bacterial release factor RF2 and initiation factor IF3 negative feedback loops are reviewed and compared with similar negative feedback loops that regulate the levels of the eukaryote release factor, eRF1, established artificially by mutation. The control properties of such negative feedback loops are discussed as well as their evolution. The role of negative feedback to control translation factor expression is considered in the context of a growing body of evidence that both IF3 and RF2 can play a role in stimulating stalled ribosomes to abandon translation in response to amino acid starvation. Here, we make the case that negative feedback control serves primarily to limit the overexpression of these translation factors, preventing the loss of fitness resulting from an unregulated increase in the frequency of ribosome drop-off.

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Section: Why Control By Negative Feedback: Translational Fidelity As mentioning

confidence: 99%

Autoregulatory systems controlling translation factor expression: Thermostat-like control of translational accuracy

Betney

Silva²,

Krishnan³

et al. 2010

RNA

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“…1A). By maintaining the same transcriptional promoter, SD region, initiation and termination codons, and the shortest possible "ORF," we attempted to minimize the number of variables affecting minigene expression and focus on the effect of the different sense codons on toxicity (9,13 Fig. 1B, showed a wide variation in the degree of toxicity to the two mutant strains arising from minigene expression and depending on the nature of the second codon: 27 were toxic to both pth(Ts) and pth(rap), 18 were lethal to pth(rap), and 16 had no deleterious effects on either mutant.…”

Section: Minigenes Harboring Codons From the Same Genetic Codementioning

confidence: 99%

“…Interestingly, a high frequency of AAA/G codons, associated to high drop-off rates in minigenes, has been found at the last sense position of the E. coli ORFs where they also promote drop-off (13). This non-random codon distribution at the ends of ORFs has been considered as an important factor in the modulation of translation termination (39).…”

Section: Minigenes Harboring Codons From the Same Genetic Codementioning

confidence: 99%

“…Drop-off during minigene mRNA translation is enhanced by these termination factors as well as the elongation factor EF-G (9). In vitro experiments with different synthetic minigenes have shown that the relative rates of termination and drop-off vary according to the composition of the last sense codon, the nature and context of the stop codon, and the length of the mini-ORF and that toxicity is correlated to these conditions (9,13). In addition, the strong effect of the SD sequence affects peptidyl-tRNA accu-* This work was supported by Consejo Nacional de Ciencia y Tecnología (CONACyT) Grants 28401N and 37759N (to G. G.) and O28 (ER025) (to Dr. Julio Collado Vives (Universidad Nacional Autónoma de México)) and by the Consejo del Sistema Nacional de Educación Tecnológica (COSNET).…”

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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

Cruz-Vera¹,

Hernandez-Ramon

Pérez-Zamorano

et al. 2003

Journal of Biological Chemistry

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The expression of some very short open reading frames (ORFs) in Escherichia coli results in peptidyltRNA 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-tRNA Arg-2 and (ii) the toxicity of minigenes containing CUG and UCU codons, each recognized by two different tRNAs, depended on peptidyltRNA 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.Minigenes are DNA sequences present in bacterial chromosomes that may be expressed into functionally active oligopeptides. In Escherichia coli for example, translation of a peptide encoded in a minigene present in the 23 S rRNA, turns cells erythromycin resistant (1); also, peptides containing five to eight amino acid residues encoded in the attenuator sequence of genes, which confer resistance to chloramphenicol and erythromycin (catA86, cmlA and ermC), inhibit peptidyltransferase activity in bacteria (2).Translation of two-codon minigenes located in bacteriophage lambda chromosome bar regions is lethal to cells partly defective in peptidyl-tRNA hydrolase activity, but not to wild-type bacteria (3). Translation of bar minigene mRNAs results in premature release of peptidyl-tRNAs from ribosomes (a phenomenon called "drop-off "); under limited Pth 1 activity, these peptidyl-tRNAs accumulate in the cell. It has been proposed that lethality stems from the subsequent shortage in the pool of specific tRNAs for further involvement in protein synthesis (4). Recently, evidence that seems to support this inference has been obtained for a ribosome bypassing system (5), but the alternative explanation that peptidyl-tRNAs might be toxic per se has not been ruled out (6).Translation ends at the termination codon in an mRNA, when the ribosomal peptidyl-transferase presumably hydrolyzes the ester bond between the completed polypeptide chain and the last tRNA. The termination reaction requires the concurrence of the release factors RF-1 or RF-2 (depending on the nature of the termination codon) and other factors catalyzing the release of t...

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“…For example, it has been shown that minigene overexpression in E. coli can inhibit both translation and cell growth (14,20). Inhibition occurs because cells fail to cleave the peptidyl-tRNA released from ribosomes efficiently, resulting in depletion of essential tRNA species (14,20).…”

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confidence: 99%

Overexpression of tnaC of Escherichia coli Inhibits Growth by Depleting tRNA ₂ ^Pro Availability

Gong

Gong²,

Yanofsky³

2006

J Bacteriol

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Transcription of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. Induction results from ribosome stalling after translation of tnaC, the coding region for a 24-residue leader peptide. The last sense codon of tnaC, proline codon 24 (CCU), is translated by tRNA 2 Pro . We analyzed the consequences of overexpression of tnaC from a multicopy plasmid and observed that under inducing conditions more than 60% of the tRNA 2 Pro in the cell was sequestered in ribosomes as TnaC-tRNA 2 Pro . The half-life of this TnaC-tRNA 2 Pro was shown to be 10 to 15 min under these conditions. Plasmid-mediated overexpression of tnaC, under inducing conditions, reduced cell growth rate appreciably. Increasing the tRNA 2 Pro level relieved this growth inhibition, suggesting that depletion of this tRNA was primarily responsible for the growth rate reduction. Growth inhibition was not relieved by overexpression of tRNA 1 Pro , a tRNA Pro that translates CCG, but not CCU. Replacing the Pro24CCU codon of tnaC by Pro24CCG, a Pro codon translated by tRNA 1 Pro , also led to growth rate reduction, and this reduction was relieved by overexpression of tRNA 1 Pro . These findings establish that the growth inhibition caused by tnaC overexpression during induction by tryptophan is primarily a consequence of tRNA Pro depletion, resulting from TnaC-tRNA Pro retention within stalled, translating ribosomes.

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Origins of minigene-dependent growth inhibition in bacterial cells

Cited by 51 publications

References 32 publications

Autoregulatory systems controlling translation factor expression: Thermostat-like control of translational accuracy

Autoregulatory systems controlling translation factor expression: Thermostat-like control of translational accuracy

The Rate of Peptidyl-tRNA Dissociation from the Ribosome during Minigene Expression Depends on the Nature of the Last Decoding Interaction

Overexpression of tnaC of Escherichia coli Inhibits Growth by Depleting tRNA ₂ ^Pro Availability

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Origins of minigene-dependent growth inhibition in bacterial cells

Cited by 51 publications

References 32 publications

Autoregulatory systems controlling translation factor expression: Thermostat-like control of translational accuracy

Autoregulatory systems controlling translation factor expression: Thermostat-like control of translational accuracy

The Rate of Peptidyl-tRNA Dissociation from the Ribosome during Minigene Expression Depends on the Nature of the Last Decoding Interaction

Overexpression of tnaC of Escherichia coli Inhibits Growth by Depleting tRNA 2 Pro Availability

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Overexpression of tnaC of Escherichia coli Inhibits Growth by Depleting tRNA ₂ ^Pro Availability