Suppression

Steege, Deborah A.; Söll, Dieter

doi:10.1007/978-1-4684-3417-0_11

Cited by 32 publications

(12 citation statements)

References 253 publications

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“…The sequence of events at peptide chain termination in eukaryotes (reviews [9,10]) is still sketchy. When a ribosome reaches a termination codon, a peptidyl-tRNA sits at the 'P site', and the 'A site' is free.…”

Section: 'Bona Fide' Terminationmentioning

confidence: 99%

Stop making sense or Regulation at the level of termination in eukaryotic protein synthesis

Valle

Morch

1988

FEBS Letters

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Section: 'Bona Fide' Terminationmentioning

confidence: 99%

Stop making sense or Regulation at the level of termination in eukaryotic protein synthesis

Valle

Morch

1988

FEBS Letters

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“…First identified in 1965 (4, 7), they have been widely used in studies of translation, phage biology, and protein engineering. They have been critical to our understanding of the structure and function, processing, and charging of tRNAs, as well as ribosome-tRNA interaction, polarity, codon context effects, and the elucidation of the genetic code (29). Suppressors have also been used to study the impact of single amino acid substitutions and nonstandard amino acids on protein function (15,30).…”

mentioning

confidence: 99%

Global Transcriptional Effects of a Suppressor tRNA and the Inactivation of the Regulator frmR

Herring

Blattner

2004

J Bacteriol

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Expression of an amber suppressor tRNA should result in read-through of the 326 open reading frames (ORFs) that terminate with amber stop codons in the Escherichia coli genome, including six pseudogenes. Abnormal extension of an ORF might alter the activities of the protein and have effects on cellular physiology, while suppression of a pseudogene could lead to a gain of function. We used oligonucleotide microarrays to determine if any effects were apparent at the level of transcription in glucose minimal medium. Surprisingly, only eight genes had significantly different expression in the presence of the suppressor. Among these were the genes yaiN, adhC, and yaiM, forming a single putative operon whose likely function is the degradation of formaldehyde. Expression of wild-type yaiN was shown to result in repression of the operon, while a suppression-mimicking allele lacking the amber stop codon and extended 7 amino acids did not. The operon was shown to be induced by formaldehyde, and the genes have been renamed frmR, frmA, and frmB, respectively.A suppressor tRNA is a tRNA with a mutation (usually) in the anticodon that allows it to recognize a stop codon and insert an amino acid in its place (reviewed in reference 6). First identified in 1965 (4, 7), they have been widely used in studies of translation, phage biology, and protein engineering. They have been critical to our understanding of the structure and function, processing, and charging of tRNAs, as well as ribosome-tRNA interaction, polarity, codon context effects, and the elucidation of the genetic code (29). Suppressors have also been used to study the impact of single amino acid substitutions and nonstandard amino acids on protein function (15,30). Amber suppressors have been used preferentially because amber (UAG) is the least common of the three stop codons, occurring at the end of 326 out of 4,290 open reading frames (ORFs) in the Escherichia coli genome. In contrast, ochre (UAA) occurs 2,706 times and opal (UGA) occurs 1,258 times. The reasons for this dramatic skew are not known, though it is interesting that 1.5% of fecal coliform isolates (mostly E. coli) were found to contain natural amber suppressors (18). Out of 13 pseudogenes in strain K-12 (identified from the EcoGene website [26] at http://bmb.med.miami.edu/EcoGene/EcoWeb/), 6 are disrupted with amber stop codons (glvC, ybfH, yehQ, yfcU, yjiP, and b2650) and almost all are of unknown function.For the functional characterization of the E. coli genome, we have developed methods to systematically introduce amber mutations into the chromosome and have constructed an arabinose-inducible suppressor tRNA on a high-copy-number plasmid to modulate the effects of the amber mutations (12, 13). To use this system it was important to examine the effects of the suppressor on the cell. In addition to suppressing the gene of interest, the suppressor is also expected to affect genes throughout the genome that end with amber stop codons. Reflecting the relative prevalence of amber and ochre stop codons, ...

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“…Both chromosomally and extra-chromosomally encoded elongator tRNA suppressors are important tools in various genetic applications (Steege & Soll, 1979). Because of their usefulness, E. coli strains (supD, supE, supF and supP) inserting serine, glutamine, tyrosine and leucine, respectively, in response to UAG codons have been generated.…”

Section: Discussionmentioning

confidence: 99%

Acquisition of a stable mutation in metY allows efficient initiation from an amber codon in Escherichia coli

et al. 2005

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Escherichia coli strains harbouring elongator tRNAs that insert amino acids in response to a termination codon during elongation have been generated for various applications. Additionally, it was shown that expression of an initiator tRNA containing a CUA anticodon from a multicopy plasmid in E. coli resulted in initiation from an amber codon. Even though the initiation-based system remedies toxicity-related drawbacks, its usefulness has remained limited for want of a strain with a chromosomally encoded initiator tRNA ‘suppressor’. E. coli K strains possess four initiator tRNA genes: the metZ, metW and metV genes, located at a single locus, encode tRNA1 fMet, and a distantly located metY gene encodes a variant, tRNA2 fMet. In this study, a stable strain of E. coli K-12 that affords efficient initiation from an amber initiation codon was isolated. Genetic analysis revealed that the metY gene in this strain acquired mutations to encode tRNA2 fMet with a CUA anticodon (a U35A36 mutation). The acquisition of the mutations depended on the presence of a plasmid-borne copy of the mutant metY and recA + host background. The mutations were observed when the plasmid-borne gene encoded tRNA2 fMet (U35A36) with additional changes in the acceptor stem (G72; G72G73) but not in the anticodon stem (U29C30A31/U35A36/ψ39G40A41). The usefulness of this strain, and a possible role for multiple tRNA1 fMet genes in E. coli in safeguarding their intactness, are discussed.

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Suppression

Cited by 32 publications

References 253 publications

Stop making sense or Regulation at the level of termination in eukaryotic protein synthesis

Stop making sense or Regulation at the level of termination in eukaryotic protein synthesis

Global Transcriptional Effects of a Suppressor tRNA and the Inactivation of the Regulator frmR

Acquisition of a stable mutation in metY allows efficient initiation from an amber codon in Escherichia coli

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