TSRI Scientists Create New Strain of Yeast with 21-Amino
Acid Genetic Code
By Jason Socrates
Henry Ford revolutionized personal transportation by introducing
an unusual car design onto the auto market and by embracing
factory mass production of his "Tin Lizzie."
Now a team of investigators at The Scripps Research Institute
(TSRI) and its Skaggs Institute for Chemical Biology in La
Jolla, California is introducing revolutionary changes into
the genetic code of organisms like yeast that allow these
cellular factories to mass produce proteins with unnatural
Led by Professor Peter G. Schultz, who holds the Scripps
Family Chair in Chemistry at TSRI, the team is reporting in
the latest issue of the journal Science a general method
for adding unnatural amino acids to the genetic code of a
type of yeast called Saccharomyces cerevisiae.
In the paper, the TSRI team describes how they incorporated
five unnatural amino acids into the yeast, a "eukaryotic"
organism that has cells with membrane-bound nuclei. Earlier
studies by the same group incorporated unnatural amino acids
in "prokaryotic" bacterial cells, which lack membrane-bound
nuclei. By demonstrating that it is possible to add unnatural
amino acids to the genetic code of yeast, the TSRI team has
set the stage for a whole new approach to applying the same
technology to other eukaryotic cells, and even multicellular
"Yeast is the gateway to mammalian cells," says Schultz.
"We've opened up the whole pathway to higher organisms."
Why Expand the Genetic Code?
Life as we know it is composed, at the molecular level,
of the same basic building blocks. For instance, all life
forms on earth use the same four nucleotides to make DNA.
And almost without exception, all known forms of life use
the same common 20 amino acidsand only those 20to
The question is why did life stop with 20 and why these
While the answer to that question may be elusive, the 20-amino
acid barrier is far from absolute. In some rare instances,
in fact, certain organisms have evolved the ability to use
the unusual amino acids selenocysteine and pyrrolysineslightly
modified versions of the amino acids cysteine and lysine.
These rare exceptions aside, scientists have often looked
for ways to incorporate other unusual amino acids into proteins
because such technologies are of great utility for basic biomedical
research. For example, there are novel amino acids that contain
fluorescent groups that can be used to site-specifically label
proteins with small fluorescent tags and observe them in vivo.
This is particularly useful now that the human genome has
been solved and scientists are now turning their attention
to what these genes are doing inside cells.
Other unnatural amino acids contain photoaffinity labels
and other "crosslinkers" that could be used for trapping proteinprotein
interactions by forcing interacting proteins to be covalently
attached to one another. Purifying these linked proteins would
allow scientists to see what proteins interact within living
cellseven those with weak interactions that are difficult
to detect by current methods.
"The more you can control proteins in the cell, the more
information you can get about what they are really doing in
their natural environment," says Jason Chin, who is the lead
author of the study.
Unnatural amino acids are also important in medicine, and
many proteins used therapeutically need to be modified with
chemical groups such as polymers, crosslinking agents, and
cytotoxic molecules. Earlier this year Schultz and his TSRI
colleagues also showed that glycosylated amino acids could
be incorporated site-specifically to make glycosylated proteinsan
important step in the preparation of some medicines.
Novel hydrophobic amino acids, heavy metal-binding amino
acids, and amino acids that contain spin labels could be useful
for probing the structures of proteins into which they are
inserted. And unusual amino acids that contain chemical moieties
like "keto" groups, which are like LEGO® blocks, could
be used to attach other chemicals such as sugar molecules,
which would be relevant to the production of therapeutic proteins.
The five amino acids that Schultz and his colleagues inserted
into the genetic code of yeast include a "benzophenone" amino
acid that can be used as a photocrosslinker; a photocrosslinker
known as an azide; a "ketone" amino acid that is like a hook
to which other molecules, such as dyes, can be attached; an
"iodo" compound that contains a heavy metal atom, which is
useful for x-ray crystallography, and the amino acid, O-methyl-tyrosin,
derivatives of which can be used in nuclear magnetic resonance
Scientists have for years created proteins with such unnatural
amino acids in the laboratory, but until Schultz and his colleagues
began their work in this field, nobody had ever found a way
to get organisms to add unnatural amino acids into their genetic
The Basis of the Technology
Schultz and his colleagues succeeded in making the 21-amino
acid yeast by exploiting the redundancy of the genetic code.
When a protein is expressed, an enzyme reads the DNA bases
of a gene (A, G, C, and T), and transcribes them into RNA
(A, G, C, and U). This so-called "messenger RNA" is then translated
by another protein-RNA complex, called the ribosome, into
a protein. The ribosome requires the help of transfer RNA
molecules (tRNA) that have been "loaded" with an amino acid,
and that requires the help of a "loading" enzyme.
Each tRNA recognizes one specific three-base combination,
or "codon," on the mRNA and gets loaded with only the one
amino acid that is specific for that codon.
During protein synthesis, the tRNA specific for the next
codon on the mRNA comes in loaded with the right amino acid,
and the ribosome grabs the amino acid and attaches it to the
growing protein chain.
The redundancy of the genetic code comes from the fact that
there are more codons than there are amino acids used. In
fact, there are 4x4x4 = 64 different possible ways to make
a codonor any three-digit combination of four letters
in the mRNA (UAG, ACG, UTC, etc.). With only 20 amino acids
used by the organisms, not all of the codons are theoretically
But nature uses them anyway. Several of the 64 codons are
redundant, coding for the same amino acid, and three of them
are nonsense codonsthey don't code for any amino acid
at all. These nonsense codons are useful because normally
when a ribosome that is synthesizing a protein reaches a nonsense
codon, the ribosome dissociates from the mRNA and synthesis
stops. Hence, nonsense codons are also referred to as "stop"
codons. One of these, UAG, played an important role in Schultz's
Schultz and his colleagues knew that if they could provide
their cells with a tRNA molecule that recognizes UAG and also
provide them with a synthetase "loading" enzyme that loaded
the tRNA with an unusual amino acid, the scientists would
have a way to site-specifically insert the unusual amino acid
into any protein they wanted.
They needed to find a functionally "orthogonal" paira
tRNA/synthetase pair that react with each other but not with
endogenous yeast pairs. They then devised a methodology to
evolve the specificity of the orthogonal synthetase to selectively
accept unnatural amino acids. They created a library of yeast
cells, each encoding a mutant synthetase, and devised a positive
selection whereby only the cells that load the orthogonal
tRNA with any amino acid would survive. Then they designed
a negative selection whereby any cell that recognizes UAG
using a tRNA loaded with a natural amino acid dies. In so
doing, they found their orthogonal synthetase mutants that
load the orthogonal tRNA with only the desired unnatural amino
With this system, a ribosome that was reading an mRNA would
insert the unusual amino acid when it encountered UAG. Furthermore,
any codon in an mRNA that is switched to UAG will encode for
the new amino acid in that place, giving Schultz and his colleagues
a way to site-specifically incorporate novel amino acids into
proteins expressed by the yeast.
"The ability to put unnatural amino acids into proteins
is an incredibly powerful tool," says Schultz.
The article, "An Expanded Eukaryotic Genetic Code" was authored
by Jason W. Chin, T. Ashton Cropp, J. Christopher Anderson,
Mridul Mukherji, Zhiwen Zhang, and Peter G. Schultz and appears
in the August 15, 2003 issue of the journal Science.