An estimate of the frequency of in vivo transcriptional errors at a nonsense codon in Escherichia coli

Rosenberger, R. F.; Foskett, Gillian

doi:10.1007/bf00268784

Cited by 66 publications

(48 citation statements)

References 25 publications

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“…S4) in B. subtilis with those obtained using an ocher nonsense codon in E. coli (15,17) revealed that our measurements are at least 10-fold higher than previous estimates, raising the possibility that an error occurs approximately once every 200 codons. Errors exceeding 0.1% have been previously reported for specific missense mutations and are generally considered exceptional (e.g.…”

Section: Si Materials and Methods)contrasting

confidence: 67%

“…In vivo measurements of errors in gene expression in bacteria have been estimated to occur at a rate of 10 −4 to 10 −5 per nucleotide during transcription and ≈10 −3 to 10 −4 per codon during translation. These error rates are significantly higher than that attributed to the highfidelity replication process, where the frequency of errors is as low as 10 −8 to 10 −9 per nucleotide (8,9,(12)(13)(14)(15)(16)(17)(18)(19)(20).…”

mentioning

confidence: 85%

“…1C). These measurements could even be underestimates, as mutations situated at the beginning of an ORF are predicted to minimize the error level as they predispose ribosomes to fall off the transcript (17,23). To further examine if the error rates detected are exceptional or reflect a more standard level, we measured the fluorescence emanated from additional gfp mutated alleles.…”

Section: Si Materials and Methods)mentioning

confidence: 99%

“…For the most part, in vivo error rates in bacterial gene expression have been estimated by measuring the activity of a mutated reporter gene (such as lacZ) (14,17,18) in a large cell population. However, the resultant measurements represent the average error rates, and thus do not detect the actual error level per cell and the variability, if any, among individuals.…”

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confidence: 99%

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Visualizing high error levels during gene expression in living bacterial cells

Meyerovich

Mamou

Ben-Yehuda

2010

Proc. Natl. Acad. Sci. U.S.A.

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To monitor inaccuracy in gene expression in living cells, we designed an experimental system in the bacterium Bacillus subtilis whereby spontaneous errors can be visualized and quantified at a single-cell level. Our strategy was to introduce mutations into a chromosomally encoded gfp allele, such that errors in protein production are reported in real time by the formation of fluorescent GFP molecules. The data reveal that the amount of errors can greatly exceed previous estimates, and that the error rate increases dramatically at lower temperatures and during stationary phase. Furthermore, we demonstrate that when facing an antibiotic threat, an increase in error level is sufficient to allow survival of bacteria carrying a mutated antibiotic-resistance gene. We propose that bacterial gene expression is error prone, frequently yielding protein molecules that differ slightly from the sequence specified by their DNA, thus generating a cellular reservoir of nonidentical protein molecules. This variation may be a key factor in increasing bacterial fitness, expanding the capability of an isogenic population to face environmental challenges.Bacillus subtilis | translational errors | variations in living cells | translation fidelity D NA is duplicated with remarkable fidelity to ensure that accurate genetic information is transmitted from one generation to the next. This information is passed from DNA to RNA and from RNA to protein during gene expression; however, the accuracy of these downstream events is relatively less understood. Although RNA and proteins are generally considered short-lived noninherited molecules, several diseases are now known to arise from errors occurring during transcription and translation (1-3), implying that faithful transfer of genetic information from DNA to proteins is crucial for maintaining proper cellular functions.

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Section: Si Materials and Methods)contrasting

confidence: 67%

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confidence: 85%

Section: Si Materials and Methods)mentioning

confidence: 99%

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confidence: 99%

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Visualizing high error levels during gene expression in living bacterial cells

Meyerovich

Mamou

Ben-Yehuda

2010

Proc. Natl. Acad. Sci. U.S.A.

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“…30 Such insertion causes a -1 frameshift, which would abolish expression of the full-length TK required to phosphorylate the ACV in order to activate its anti-viral activity. 31 On the RNA level, although the transcription error rate is considered much lower than those during DNA replication, 32 insertion or deletion of a nucleotide can also occur during transcription leading to a frameshift. 33,34,35 The insertion/deletion is believed to involve a slippage between the DNA and RNA strands similar to the Streisinger DNA slippage model, and also tends to occur more frequently on mononucleotide repeats.…”

Section: Discussionmentioning

confidence: 99%

Identification and characterization of a -1 reading frameshift in the heavy chain constant region of an IgG1 recombinant monoclonal antibody produced in CHO cells

Lian

Wang

2015

mAbs

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Frameshifts lead to complete alteration of the intended amino acid sequences, and therefore may affect the biological activities of protein therapeutics and pose potential immunogenicity risks. We report here the identification and characterization of a novel -1 frameshift variant in a recombinant IgG1 therapeutic monoclonal antibody (mAb) produced in Chinese hamster ovary cells during the cell line selection studies. The variant was initially observed as an atypical post-monomer fragment peak in size exclusion chromatography. Characterization of the fragment peak using intact and reduced liquid chromatographymass spectrometry (LC-MS) analyses determined that the fragment consisted of a normal light chain disulfide-linked to an aberrant 26 kDa fragment that could not be assigned to any HC fragment even after considering common modifications. Further analysis using LC-MS/MS peptide mapping revealed that the aberrant fragment contained the expected HC amino acid sequence (1-232) followed by a 20-mer novel sequence corresponding to expression of heavy chain DNA sequence in the -1 reading frame. Examination of the DNA sequence around the frameshift initiation site revealed that a mononucleotide repeat GGGGGG located in the IgG1 HC constant region was most likely the structural root cause of the frameshift. Rapid identification of the frameshift allowed us to avoid use of a problematic cell line containing the frameshift as the production cell line. The frameshift reported here may be observed in other mAb products and the hypothesis-driven analytical approaches employed here may be valuable for rapid identification and characterization of frameshift variants in other recombinant proteins.

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Fehlerhäufigkeit bei der Replikation und Expression der genetischen Information

et al. 1985

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Die biologische Evolution wird nur durch standige Veranderungen in den Genomen der Zellen ermoglicht. Da trotzdem bei diesem ProzeD stabile Organismen entstehen, mu13 eine niedrige Fehlerhaufigkeit oder anders ausgedriickt eine hohe Genauigkeit bei der Weitergabe der genetischen Information und ihrer Umsetzung in Proteinsequenzen gewahrleistet sein. Die Fehlerhaufigkeit dieses gesamten Vorgangs kann man messen und rechnerisch abschatzen: Sie ergibt sich aus den Fehlerhaufigkeiten der enzymatisch katalysierten Einzelschritte. In vielen Fallen, z. B. bei der Erkennung von Valin und Isoleucin wahrend der Proteinbiosynthese, ist eine ausreichende Genauigkeit nicht allein aufgrund der unterschiedlichen Bindungsenergien des richtigen und des falschen Substrats zu erzielen (die beiden Aminosauren unterscheiden sich nur durch eine CH2-Gruppe). Die erforderliche niedrige Fehlerhaufigkeit wird durch einen zusatzlichen Priif-und gegebenenfalls Korrekturschritt erreicht, den die beteiligten Enzyme (nach Bildung des Enzym-Substrat-Komplexes wahrend oder nach Ablauf des katalytischen Schrittes) ausfuhren; dabei wird eine als nicht richtig erkannte Zwischenstufe oder ein als nicht richtig erkanntes Produkt hydrolytisch gespalten. Die Energie zur Synthese des falschen Produkts, die durch Hydrolyse von Adenosin-oder Guanosintriphosphat aufgebracht wird, geht als Preis ftir die niedrige Fehlerhaufigkeit verloren. In diesem Beitrag sollen -hauptsachlich an den Beispielen der DNA-Replikation und der Aminoacylierung der Transfer-RNA -die Priif-und Korrekturmechanismen von einigen Enzymen vorgestellt werden.

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An estimate of the frequency of in vivo transcriptional errors at a nonsense codon in Escherichia coli

Cited by 66 publications

References 25 publications

Visualizing high error levels during gene expression in living bacterial cells

Visualizing high error levels during gene expression in living bacterial cells

Identification and characterization of a -1 reading frameshift in the heavy chain constant region of an IgG1 recombinant monoclonal antibody produced in CHO cells

Fehlerhäufigkeit bei der Replikation und Expression der genetischen Information

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