Codon context effects on nonsense suppression in human cells

Martin, Robert H.; Phillips‐Jones, Mary K.; Watson, Fiona; Hill, Lindsey S. J.

doi:10.1042/bst0210846

Cited by 20 publications

(26 citation statements)

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“…In our previous study of nonsense suppression context effects at an N-terminally located UAG codon (40) it was difficult to study the context sensitivity of a second suppressor, tRNA Gln UAG (28), because this suppressor is substantially weaker than tRNA Ser UAG (14, 34a). Consequently, the activity from tRNA Gln UAG was insufficient to enable a clear distinction to be made between suppression and translational reinitiation downstream from the N-terminal UAG (36). Very little background activity could be detected at the new site in the present study, and so, it proved possible to study 3Ј codon context effects in human cells on a second suppressor tRNA.…”

Section: Effect Of 3 Codon Context On Nonsense Suppression By Trnamentioning

confidence: 85%

“…This construction was advantageous for studies in which nucleotides 3Ј to the nonsense codon were altered to test their effect on translation through the stop codon, since changes to the flanking amino acids cannot interfere with the enzymatic signal from the ␤-galactosidase reporter. A disadvantage of these vectors, however, was the level of unsuppressed background caused by ribosome reinitiation (36,40). At 2 to 4% of the wild-type level, this was sufficient to obscure measurements of nonsense suppression by a weak suppressor and misreading induced by the aminoglycoside G418.…”

Section: Construction Of Prsvgal Reporters Containing Uag In Differenmentioning

confidence: 99%

“…The pRSV559 series contain UAG at lacZ codon 600 followed by either A, C, G, or U. The lacZ gene in pRSVgal is an N-terminal translational fusion, gpt-trpS-lacZ, in which lacZ codon 600 is in fact codon 662 (36,40). The pRSV600 series contain the wild-type CAG (Gln) codon, again with either an A, a C, a G, or a U as a 3Ј context.…”

Section: Methodsmentioning

confidence: 99%

“…In the face of a background of translational reinitiation it was not possible to detect any G418-induced misreading at N-terminal UAG codons (36,40). G418-mediated suppression was tested in the present study at the 559 UAG mutation in each 3Ј context.…”

Section: G418-induced Misreading At Uag Codons In Human 293 Cellsmentioning

confidence: 99%

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Context Effects on Misreading and Suppression at UAG Codons in Human Cells

Phillips‐Jones

Hill

Atkinson

et al. 1995

Molecular and Cellular Biology

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The effect of the 3 codon context on the efficiency of nonsense suppression in mammalian tissue culture cells has been tested. Measurements were made following the transfection of cells with a pRSVgal reporter vector that contained the classical Escherichia coli lacZ UAG allele YA559. The position of this mutation was mapped by virtue of its fortuitous creation of a CTAG MaeI restriction enzyme site. Determination of the local DNA sequence revealed a C3T mutation at codon 600 of the lacZ gene: CAG3TAG. Site-directed mutagenesis was used to create a series of vectors in which the base 3 to the nonsense codon was either A, C, G, or U. Suppression of the amber-containing reporter was achieved by cotransfection with genes for human tRNA Ser or tRNA Gln UAG nonsense suppressors and by growth in the translational error-promoting aminoglycoside drug G418. Nonsense suppression was studied in the human cell lines 293 and MRC5V1 and the simian line COS-7. Overall, the rank order for the effect of changes to the base 3 to UAG was C < G ‫؍‬ U < A. This study confirms and extends earlier findings that in mammalian cells 3 C supports efficient nonsense suppression while 3 A is unsympathetic for read-through at nonsense codons. The rules for the mammalian codon context effect on nonsense suppression are therefore demonstrably different from those in E. coli.Normally, nonsense or stop codons catalyze the effective cessation of protein synthesis. However, when tRNA species within the cell are able to recognize a nonsense codon, the function of the stop is suppressed. Nonsense suppression has been exploited as a means for the conditional expression of genes containing stop codons in order to study their function (23,44). Nonsense suppression has also been used extensively in Escherichia coli as a tool for monitoring the efficiency of translation at an individual codon (46,47,51). Of particular interest is the facility that nonsense suppression affords for determining the effects of 5Ј and 3Ј contexts on the interaction of translation factors and aminoacyl-tRNAs with their mRNA targets (19,39,45,49,52). Recently, nonsense suppression has been harnessed in order to carry out in vitro protein engineering. In this process, chemically acylated nonsense suppressor tRNAs are used to insert nonnatural amino acid residues at specified locations by targeting a nonsense codon which interrupts the coding sequence (2, 3, 33).There have been comparatively few reports of nonsense suppression in mammalian cells, but the transfection of sensitively detected reporters now allows this technique to be applied to cells in culture (6,12,13,15,43). In a previous report from this laboratory, we described the effects of alterations in the 3Ј mRNA context on the efficiency of a human tRNA Ser UAG nonsense suppressor (40). The function of the suppressor was monitored by determining the efficiency of translation at a UAG codon in a ␤-galactosidase reporter gene which had been cotransfected into human 293 cells with a plasmid vector expressing a human UAG su...

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Section: Effect Of 3 Codon Context On Nonsense Suppression By Trnamentioning

confidence: 85%

Section: Construction Of Prsvgal Reporters Containing Uag In Differenmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: G418-induced Misreading At Uag Codons In Human 293 Cellsmentioning

confidence: 99%

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Context Effects on Misreading and Suppression at UAG Codons in Human Cells

Phillips‐Jones

Hill

Atkinson

et al. 1995

Molecular and Cellular Biology

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“…For example, the first nucleotide following the codon (2), the second nucleotide following (3), the third nucleotide following, and the preceding codon (1,4) all modulate suppression. Less information is available for mammalian contexts, but a study of suppression at UAG codons in human cells demonstrated that the 3' nucleotide influenced the efficiency of suppression (C > G > U = A) in a different order from that of Escherichia coli (A > G > C > U) (5). Translational readthrough at a stop codon in the gene encoding the heat shock transcription factor from Saccharomyces cerevisiae was shown to occur when the stop codon was followed by A or C but not when it was followed by G (6).…”

Section: Introductionmentioning

confidence: 99%

Translational termination efficiency in mammals is influenced by the base following the stop codon.

McCaughan

Brown

Dalphin

et al. 1995

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

269

190

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The base following stop codons in mammalian genes is strongly biased, suggesting that it might be important for the termination event. This proposal has been tested experimentally both in vivo by using the human type I iodothyronine deiodinase mRNA and the recoding event at the internal UGA codon and in vitro by measuring the ability of each of the 12 possible 4-base stop signals to direct the eukaryotic polypeptide release factor to release a model peptide, formylmethionine, from the ribosome. The internal UGA in the deiodinase mRNA is used as a codon for incorporation of selenocysteine into the protein. Changing the base following this UGA codon affected the ratio of termination to selenocysteine incorporation in vivo at this codon: 1:3 (C or U) and 3:1 (A or G). These UGAN sequences have the same order of efficiency of termination as was found with the in vitro termination assay (4th base: A G> > C U). The efficiency of in vitro termination varied in the same manner over a 70-fold range for the UAAN series and over an 8-fold range for the UGAN and UAGN series. There is a correlation between the strength of the signals and how frequently they occur at natural termination sites. Together these data suggest that the base following the stop codon influences translational termination efficiency as part of a larger termination signal in the expression of mammalian genes.

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