Shutdown in protein synthesis due to the expression of mini-genes in bacteria

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

doi:10.1006/jmbi.1999.3028

Cited by 55 publications

(94 citation statements)

References 32 publications

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“…Box Show Similar Toxicity-Changes in the last sense codon of some minigenes determine variations in the degree of toxicity for pth mutants (9,24,25). To understand the nature of this effect, we used an expression library of two-codon ORF minigenes in which the second, and last, sense codon was any of the 64 possibilities in the genetic code (Fig.…”

Section: Minigenes Harboring Codons From the Same Genetic Codementioning

confidence: 99%

“…: 52-55-747-3338; Fax: 52-55-747-7100; E-mail: guarnero@lambda.gene.cinvestav.mx. mulation by driving minigene mRNA through several rounds of translation without dissociation from the ribosome (9). Despite these results, the scarce number and heterogeneity of minigenes studied so far does not allow us to draw conclusions about the role of codon composition in drop-off.…”

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confidence: 99%

“…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 the mature protein (7,8). Drop-off is a normal, if relatively rare, event in protein synthesis that can occur during elongation or instead of polypeptide termination (9,10). If the rates of peptidyl-tRNA synthesis and drop-off exceed the rates of termination and Pth hydrolysis, peptidyl-tRNA accumulates and thus critically reduces the concentration of aminoacylable tRNA and increases that of peptidyl-tRNAs (4,9).…”

mentioning

confidence: 99%

“…Drop-off is a normal, if relatively rare, event in protein synthesis that can occur during elongation or instead of polypeptide termination (9,10). If the rates of peptidyl-tRNA synthesis and drop-off exceed the rates of termination and Pth hydrolysis, peptidyl-tRNA accumulates and thus critically reduces the concentration of aminoacylable tRNA and increases that of peptidyl-tRNAs (4,9). The up-shift to non-permissive temperatures of a thermosensitive pth mutant, pth(Ts), results in peptidyl-tRNA buildup of all the tRNAs assayed.…”

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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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confidence: 99%

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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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Section: Minigenes Harboring Codons From the Same Genetic Codementioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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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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Heterogeneity of single molecule FRET signals reveals multiple active ribosome subpopulations

Wang

Xiao

2013

Proteins

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Single molecule methods have revealed that heterogeneity is common in biological systems. However, interpretations of the complex signals are challenging. By tracking the fluorescence resonance energy transfer (FRET) signals between the A-site tRNA and L27 protein in single ribosomes, we attempt to develop a qualitative method to subtract the inherent patterns of the heterogeneous single molecule FRET data. Seven ribosome subpopulations are identified using this method and spontaneous exchanges among these subpopulations are observed. All of the pretranslocation subpopulations are competent in real-time translocation, but via distinguished pathways. These observations suggest that the ribosome may function through multiple reaction pathways.

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Distinct Stages of Protein Evolution as Suggested by Protein Sequence Analysis

Trifonov

Kirzhner

et al. 2001

Journal of Molecular Evolution

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Evolution of proteins encoded in nucleotide sequences began with the advent of the triplet code. The chronological order of the appearance of amino acids on the evolution scene and the steps in the evolution of the triplet code have been recently reconstructed (Trifonov, 2000b) on the basis of 40 different ranking criteria and hypotheses. According to the consensus chronology, the pair of complementary GGC and GCC codons for the amino acids alanine and glycine appeared first. Other codons appeared as complementary pairs as well, which divided their respective amino acids into two alphabets, encoded by triplets with either central purines or central pyrimidines: G, D, S, E, N, R, K, Q, C, H, Y, and W (Glycine alphabet G) and A, V, P, S, L, T, I, F, and M (Alanine alphabet A). It is speculated that the earliest polypeptide chains were very short, presumably of uniform length, belonging to two alphabet types encoded in the two complementary strands of the earliest mRNA duplexes. After the fusion of the minigenes, a mosaic of the alphabets would form. Traces of the predicted mosaic structure have been, indeed, detected in the protein sequences of complete prokaryotic genomes in the form of weak oscillations with the period 12 residues in the form of alteration of two types of 6 residue long units. The next stage of protein evolution corresponded to the closure of the chains in the loops of the size 25-30 residues (Berezovsky et al., 2000). Autocorrelation analysis of proteins of 23 complete archaebacterial and eubacterial genomes revealed that the preferred distances between valine, alanine, glycine, leucine, and isoleucine along the sequences are in the same range of 25-30 residues, indicating that the loops are primarily closed by hydrophobic interactions between the ends of the loops. The loop closure stage is followed by the formation of typical folds of 100-200 amino acids, via end-to-end fusion of the genes encoding the loop-size chains. This size was apparently dictated by the optimal ring closure for DNA. In both cases the closure into the ring (loop) rendered evolutionarily advantageous stability to the respective structures. Further gene fusions lead to the formation of modern multidomain proteins. Recombinational gene splicing is likely to have appeared after the DNA circularization stage.

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Shutdown in protein synthesis due to the expression of mini-genes in bacteria

Cited by 55 publications

References 32 publications

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

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

Heterogeneity of single molecule FRET signals reveals multiple active ribosome subpopulations

Distinct Stages of Protein Evolution as Suggested by Protein Sequence Analysis

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