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Increased gene translation stringency in mammalian cells by nonsense suppression at multiple permissive sites with a single noncanonical amino acid
Author(s) -
Kadunc Lucija,
Svetličič Maja,
Forstnerič Vida,
Hafner Bratkovič Iva,
Jerala Roman
Publication year - 2020
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1002/1873-3468.13810
Subject(s) - translation (biology) , amino acid , gene , nonsense mutation , nonsense mediated decay , biology , stop codon , transfer rna , nonsense , protein biosynthesis , eukaryotic translation , gene expression , microbiology and biotechnology , genetics , messenger rna , biochemistry , mutation , rna , rna splicing , missense mutation
The considerable potential of engineered cells compels the development of strategies for the stringent control of gene expression. A promising approach is the introduction of a premature stop codon (PTC) into a selected gene that is expressed only in the presence of noncanonical amino acids through nonsense suppression. Here, different strategies of amber PTC readthrough in mammalian cells were tested. The use of a tRNA synthetase together with a TAG codon‐specific tRNA achieved PTC readthrough depending on the addition of a noncanonical amino acid (4‐benzoyl‐ L ‐phenylalanine; Bpa). While single TAG codon incorporation exhibited detectable expression of the reporter protein even in the absence of Bpa, the use of a double PTC enabled virtually leakage‐free functional gene translation. The introduction of an additional 5′‐PTC, therefore, represents a generally applicable strategy to increase stringency in gene translation.