Import of amber and ochre suppressor tRNAs into mammalian cells: A general approach to site-specific insertion of amino acid analogues into proteins
AUTOR(ES)
Köhrer, Caroline
FONTE
The National Academy of Sciences
RESUMO
A general approach to site-specific insertion of amino acid analogues into proteins in vivo would be the import into cells of a suppressor tRNA aminoacylated with the analogue of choice. The analogue would be inserted at any site in the protein specified by a stop codon in the mRNA. The only requirement is that the suppressor tRNA must not be a substrate for any of the cellular aminoacyl–tRNA synthetases. Here, we describe conditions for the import of amber and ochre suppressor tRNAs derived from Escherichia coli initiator tRNA into mammalian COS1 cells, and we present evidence for their activity in the specific suppression of amber (UAG) and ochre (UAA) codons, respectively. We show that an aminoacylated amber suppressor tRNA (supF) derived from the E. coli tyrosine tRNA can be imported into COS1 cells and acts as a suppressor of amber codons, whereas the same suppressor tRNA imported without prior aminoacylation does not, suggesting that the supF tRNA is not a substrate for any mammalian aminoacyl–tRNA synthetase. These results open the possibility of using the supF tRNA aminoacylated with an amino acid analogue as a general approach for the site-specific insertion of amino acid analogues into proteins in mammalian cells. We discuss the possibility further of importing a mixture of amber and ochre suppressor tRNAs for the insertion of two different amino acid analogues into a protein and the potential use of suppressor tRNA import for treatment of some of the human genetic diseases caused by nonsense mutations.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=64678Documentos Relacionados
- Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells
- In vivo aminoacylation of human and Xenopus suppressor tRNAs constructed by site-specific mutagenesis.
- Novel amber suppressor tRNAs of mammalian origin.
- An amber suppressor tRNA gene derived by site-specific mutagenesis: cloning and function in mammalian cells.
- Twenty-first aminoacyl-tRNA synthetase–suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins in eukaryotes and in eubacteria