Four-base codon-mediated incorporation of nonnatural amino acids into proteins in a eukaryotic cell-free translation system

Taira, Hikaru; Fukushima, Masaharu; Hohsaka, Takahiro; Sisido, Masahiko

doi:10.1263/jbb.99.473

Cited by 26 publications

(21 citation statements)

References 15 publications

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“…Amber suppression is highly efficient when the average maximal peak current (I max ) is measured at 1.25 ng of tRNA per oocyte and decreases slightly when 2.5 ng is added (Table 2). CGGG shows lower suppression than GGGU, in agreement with previous in vitro studies (7,16). CGGG suppression is highly nonlinear, with a 330% increase in current when twice as much tRNA is injected (Table 2).…”

Section: Resultssupporting

confidence: 90%

In vivo incorporation of multiple unnatural amino acids through nonsense and frameshift suppression

Rodriguez

Lester²,

Dougherty³

2006

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

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Site-specific incorporation of unnatural amino acids (UAAs) into proteins is a valuable tool for studying structure-function relationships, incorporating biophysical probes, and elucidating protein-protein interactions. In higher eukaryotic cells, the methodology is currently limited to incorporation of a single UAA in response to a stop codon, which is known as nonsense suppression. Frameshift suppression is a unique methodology for incorporating UAAs in response to quadruplet codons, but currently, it is mostly limited to in vitro protein translation systems. Here, we evaluate the viability of frameshift suppression in Xenopus oocytes. We demonstrate UAA incorporation by using yeast phenylalanine frameshift suppressor (YFFS) tRNAs that recognize two different quadruplet codons (CGGG and GGGU) in vivo. Suppression efficiency of the YFFS tRNAs increases nonlinearly with the amount of injected tRNA, suggesting a significant competition with endogenous, triplet-recognizing tRNA. Both frameshift suppressor tRNAs are less efficient than the amber suppressor tRNA THG73 (Tetrahymena thermophila G73), which has been used extensively for UAA incorporation in Xenopus oocytes. However, the two YFFS tRNAs are more ''orthogonal'' to the Xenopus system than THG73, and they offer a viable replacement when suppressing at promiscuous sites. To illustrate the potential of combining nonsense and frameshift suppression, we have site-specifically incorporated two and three UAAs simultaneously into a neuroreceptor expressed in vivo.nicotinic receptor ͉ tRNA ͉ quadruplet codon ͉ stop codon ͉ protein engineering T he site-specific incorporation of unnatural amino acids (UAAs) into proteins biosynthetically is a powerful methodology that is seeing increasing use. The primary approach has been stop codon (nonsense) suppression using a specially designed tRNA with an anticodon that recognizes the stop codon. A wide range of in vitro translation systems has been used, along with expression in Escherichia coli and, to a lesser extent, yeast. Nonsense suppression in higher eukaryotes has, for the most part, been limited to the Xenopus oocyte, where microinjection of the required mRNA and aminoacyl tRNA is straightforward and electrophysiology provides a sensitive probe of UAA incorporation (1, 2). Other experiments in higher eukaryotes have relied on the evolution of a unique tRNA and a complementary aminoacyl-tRNA synthetase (aaRS) to insert a UAA in response to the UAG or UGA stop codon, but currently, only 3-iodo-tyrosine (3), p-benzoyl-phenylalanine (4), and 5-hydroxy-tryptophan (5) have been incorporated.A remarkable variant of this approach is the use of quadruplet codons, a process that is termed frameshift suppression and was pioneered by Sisido and coworkers (6, 7). The success of this approach opens up the possibility of developing multiple additional codons, thus incorporating several different UAAs into a protein. This multiple incorporation, in turn, would enable the use of innovative biophysical approaches such as incorporatin...

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Section: Resultssupporting

confidence: 90%

In vivo incorporation of multiple unnatural amino acids through nonsense and frameshift suppression

Rodriguez

Lester²,

Dougherty³

2006

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

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“…Because the aa-tRNA Cysderived suppressors used here are all present at equivalent concentrations, our results suggest that reduced suppression at the opal and ochre codons may also be caused by less efficient competition with translation termination factor eRF1/eRF3 (Köhrer et al 2004;Taira et al 2005;Rodriguez et al 2007). One possible explanation is that Watson-Crick codon-anticodon base-pairing with amber, as opposed to opal and ochre, tRNAs in the ribosome A site more favorably stimulate the forward reaction of eEF-1a GTP hydrolysis and peptide bond formation (Rodnina et al 2005;Youngman et al 2008).…”

Section: Discussionmentioning

confidence: 89%

“…Like amber suppressors, opal and ochre tRNAs are usually derived from a native scaffold in which the anticodon and/or adjacent bases have been mutated to pair with a UGA or UAA codon, respectively (Köhrer et al 2004). tRNA Phederived opal and ochre tRNAs have been used to incorporate nitrophenylalanine in rabbit reticulocyte lysate (RRL) (Taira et al 2005), and opal suppressors derived from tRNA Gln or tRNA Trp have been used to incorporate 5-hydroxytryptophan (Zhang et al 2004) and 5-F-tryptophan (Rodriguez et al 2007), respectively, in mammalian cells. Ochre suppressors derived from Escherichia coli supF tRNA and the suppressors of all three nonsense codons derived from E. coli tRNA Gln have also been used to incorporate tyrosine (Köhrer et al 2003) and glutamine (Köhrer et al 2004) residues in mammalian cells, respectively.…”

Section: Introductionmentioning

confidence: 99%

In vitro incorporation of nonnatural amino acids into protein using tRNA^Cys-derived opal, ochre, and amber suppressor tRNAs

Gubbens¹,

Kim²,

Yang³

et al. 2010

RNA

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Amber suppressor tRNAs are widely used to incorporate nonnatural amino acids into proteins to serve as probes of structure, environment, and function. The utility of this approach would be greatly enhanced if multiple probes could be simultaneously incorporated at different locations in the same protein without other modifications. Toward this end, we have developed amber, opal, and ochre suppressor tRNAs derived from Escherichia coli, and yeast tRNA Cys that incorporate a chemically modified cysteine residue with high selectivity at the cognate UAG, UGA, and UAA stop codons in an in vitro translation system. These synthetic tRNAs were aminoacylated in vitro, and the labile aminoacyl bond was stabilized by covalently attaching a fluorescent dye to the cysteine sulfhydryl group. Readthrough efficiency (amber > opal > ochre) was substantially improved by eRF1/eRF3 inhibition with an RNA aptamer, thus overcoming an intrinsic hierarchy in stop codon selection that limits UGA and UAA termination suppression in higher eukaryotic translation systems. This approach now allows concurrent incorporation of two different modified amino acids at amber and opal codons with a combined apparent readthrough efficiency of up to 25% when compared with the parent protein lacking a stop codon. As such, it significantly expands the possibilities for incorporating nonnative amino acids for protein structure/function studies.

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“…However, previous work has shown that frameshift suppressors derived from amber suppressor tRNAs are less efficient in rabbit reticulocyte lysate (Taira et al 2005) and in Xenopus oocytes (Rodriguez et al 2006), so we did not screen frameshift suppressor tRNAs derived from ENAS.…”

Section: Enas and Enas A71 Suppression Efficiencymentioning

confidence: 99%

“…However, when using a chemically aminoacylated tRNA, an opal suppressor that efficiently suppresses the opal codon and is adequately orthogonal is currently lacking. Sisido and colleagues tested a yeast Phe opal suppressor in rabbit reticulocyte lysate, but the suppression efficiency was only 15% (compared to 65% for the yeast Phe amber suppressor) (Taira et al 2005). An opal suppressor created by changing the anticodon of THG73 to UCA resulted in large amounts of aminoacylation in vitro (Cload et al 1996).…”

Section: Suppression Efficiency Of the Acceptor Stem Mutations Does Nmentioning

confidence: 99%

Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 2: Evaluating suppression efficiency

Rodriguez¹,

Lester²,

Dougherty³

2007

RNA

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The incorporation of unnatural amino acids into proteins is a valuable tool for addition of biophysical probes, bio-orthogonal functionalities, and photoreactive cross-linking agents, although these approaches often require quantities of protein that are difficult to access with chemically aminoacylated tRNAs. THG73 is an amber suppressor tRNA that has been used extensively, incorporating over 100 residues in 20 proteins. In vitro studies have shown that the Escherichia coli Asn amber suppressor (ENAS) suppresses better than THG73. However, we report here that ENAS suppresses with <26% of the efficiency of THG73 in Xenopus oocytes. We then tested the newly developed Tetrahymena thermophila Gln amber suppressor (TQAS) tRNA library, which contains mutations in the second to fourth positions of the acceptor stem. The acceptor stem mutations have no adverse effect on suppression efficiency and, in fact, can increase the suppression efficiency. Combining mutations causes an averaging of suppression efficiency, and increased suppression efficiency does not correlate with increased DG of the acceptor stem. We created a T. thermophila opal suppressor, TQOpS9, which shows ;50% suppression efficiency relative to THG73. The TQAS tRNA library, composed of functional suppressor tRNAs, has been created and will allow for screening in eukaryotic cells, where rapid analysis of large libraries is not feasible.

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Four-base codon-mediated incorporation of nonnatural amino acids into proteins in a eukaryotic cell-free translation system

Cited by 26 publications

References 15 publications

In vivo incorporation of multiple unnatural amino acids through nonsense and frameshift suppression

In vivo incorporation of multiple unnatural amino acids through nonsense and frameshift suppression

In vitro incorporation of nonnatural amino acids into protein using tRNA^Cys-derived opal, ochre, and amber suppressor tRNAs

Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 2: Evaluating suppression efficiency

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Four-base codon-mediated incorporation of nonnatural amino acids into proteins in a eukaryotic cell-free translation system

Cited by 26 publications

References 15 publications

In vivo incorporation of multiple unnatural amino acids through nonsense and frameshift suppression

In vivo incorporation of multiple unnatural amino acids through nonsense and frameshift suppression

In vitro incorporation of nonnatural amino acids into protein using tRNACys-derived opal, ochre, and amber suppressor tRNAs

Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 2: Evaluating suppression efficiency

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In vitro incorporation of nonnatural amino acids into protein using tRNA^Cys-derived opal, ochre, and amber suppressor tRNAs