supN ochre suppressor gene in Escherichia coli codes for tRNALys

Uemura, Hirotsugu; Thorbjarnardóttir, Sigrídur H.; Gamulin, Vera; Yano, Juliano; Andrésson, O S; Söll, Dieter; Eggertsson, Guðmundur

doi:10.1128/jb.163.3.1288-1289.1985

Cited by 16 publications

(4 citation statements)

References 16 publications

(12 reference statements)

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“…If this need for additional tRNALYS were to overcome the sequestration of the tRNA in the form of peptidyl-tRNALYS, as seemed likely, then the requirement should only be satisfied by tRNALYS able to read normal Lys codons and not by an altered tRNALYS that had lost this ability. In order to test this prediction, we made use of an ochre suppressor (supN) strain shown by Uemura et al (1985) to carry a mutation in lysV; leading to an altered anticodon sequence. The 1 kb ClaI-PstI fragment carrying the valU operon (see Figure 1) was therefore cloned from a PCR-amplified fragment into pWSK29 from either a wild-type strain or the lysV(SuUAA/G) strain GE875, yielding plasmids pVH153 and pVH154 respectively.…”

Section: Identification Of the Cloned Suppressormentioning

confidence: 99%

The growth defect in Escherichia coli deficient in peptidyl-tRNA hydrolase is due to starvation for Lys-tRNA(Lys).

et al. 1996

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The existence of a conditional lethal temperature‐sensitive mutant affecting peptidyl‐tRNA hydrolase in Escherichia coli suggests that this enzyme is essential to cell survival. We report here the isolation of both chromosomal and multicopy suppressors of this mutant in pth, the gene encoding the hydrolase. In one case, the cloned gene responsible for suppression is shown to be lysV, one of three genes encoding the unique lysine acceptor tRNA; 10 other cloned tRNA genes are without effect. Overexpression of lysV leading to a 2‐ to 3‐fold increase in tRNA(Lys) concentration overcomes the shortage of peptidyl‐tRNA hydrolase activity in the cell at non‐permissive temperature. Conversely, in pth, supN double mutants, where the tRNA(Lys) concentration is reduced due to the conversion of lysV to an ochre suppressor (supN), the thermosensitivity of the initial pth mutant becomes accentuated. Thus, cells carrying both mutations show practically no growth at 39 degrees C, a temperature at which the pth mutant grows almost normally. Growth of the double mutant is restored by the expression of lysV from a plasmid. These results indicate that the limitation of growth in mutants of E.coli deficient in Pth is due to the sequestration of tRNA(Lys) as peptidyl‐tRNA. This is consistent with previous observations that this tRNA is particularly prone to premature dissociation from the ribosome.

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Section: Identification Of the Cloned Suppressormentioning

confidence: 99%

The growth defect in Escherichia coli deficient in peptidyl-tRNA hydrolase is due to starvation for Lys-tRNA(Lys).

et al. 1996

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“…There are at least three tRNALy,U genes, giving rise to ochre suppressors supL and supN. Two of these genes, lysTa and lysTf3, are in the same operon (230), but the third gene, lysV, is at a different map position (207). (iv) Two tandemly duplicated genes, tyrT and tyrV, code for tRNATyJA1 and mutate to give supF or supC.…”

Section: Suppressors Arising By Single Base Substitutions In Trna Genesmentioning

confidence: 99%

Transfer ribonucleic acid-mediated suppression of termination codons in Escherichia coli.

Eggertsson¹,

Söll²

1988

Microbiological Reviews

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129

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“…No mutation in the plasmid DNA was observed. However, sequencing of the six copies of lysyl‐tRNA in the genome revealed a U→A mutation in the third nucleotide of the lysyl‐tRNA anticodon for the LysT gene, thereby generating a 5′‐UUA‐3′ anticodon . This mutation affords a nonsense suppressor tRNA that can insert Lys at the TAG codon by G/U wobble‐pair formation.…”

Section: Figurementioning

confidence: 99%

A Strategy for Creating Organisms Dependent on Noncanonical Amino Acids

Xuan

Schultz

2017

Angew Chem Int Ed

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The use of noncanonical amino acids (ncAAs) to control an organism's viability provides a strategy for the development of conditional ‘kill switches’ for live vaccines or engineered human cells. Here we report an approach inspired by the posttranslational acetylation/deacetylation of lysine residues, in which a protein encoded by a gene with an in-frame nonsense codon at an essential lysine can be expressed in its native state only upon genetic incorporation of AcK, and subsequent enzymatic deacetylation in the host cell. We have applied this strategy to two essential E. coli enzymes, the branched chain aminotransferase BCAT and the DNA replication initiator protein DnaA. We also devised a barnase-based conditional suicide switch to further lower the escape frequency of the host cells. This strategy offers a number of attractive features for controlling host viability, including a single small molecule-based kill switch, low escape frequency and unaffected protein function.

show abstract

supN ochre suppressor gene in Escherichia coli codes for tRNALys

Cited by 16 publications

References 16 publications

The growth defect in Escherichia coli deficient in peptidyl-tRNA hydrolase is due to starvation for Lys-tRNA(Lys).

The growth defect in Escherichia coli deficient in peptidyl-tRNA hydrolase is due to starvation for Lys-tRNA(Lys).

Transfer ribonucleic acid-mediated suppression of termination codons in Escherichia coli.

A Strategy for Creating Organisms Dependent on Noncanonical Amino Acids

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