Expanding the Genetic Code:
TSRI Scientists Synthesize 21-Amino Acid Bacterium
By Jason Socrates
Scientists at The Scripps Research Institute (TSRI) report
in an upcoming article in the Journal of the American Chemical
Society their synthesis of a form of the bacterium Escherichia
coli with a genetic code that uses 21 basic amino acid
building blocks to synthesize proteinsinstead of the
20 found in nature.
This is the first time that anyone has created a completely
autonomous organism that uses 21 amino acids and has the metabolic
machinery to build those amino acids.
"We now have the opportunity to ask whether a 21-amino acid
form of life has an evolutionary advantage over life with
20 amino acids," says the report's lead author Peter Schultz,
TSRI professor of chemistry and Scripps Family Chair of TSRI's
Skaggs Institute of Chemical Biology.
"We have effectively removed a billion-year constraint on
our ability to manipulate the structure and function of proteins,"
In addition to demonstrating that life is possible with
additional amino acids, the work is of great relevance to
science and medicine because it enables scientists to chemically
manipulate the proteins that an organism produces within the
organism itself. This gives scientists a powerful tool for
research, from determining molecular structures to creating
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," asks Schultz, "why did life stop with
20 and why these 20?"
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 medical
research. For example, many proteins used therapeutically
need to be modified with chemical groups such as polymers,
crosslinking agents and cytotoxic molecules. This technology
will also be useful in basic biomedical research. For example,
there are novel amino acids that contain fluorescent groups
that can be used to label proteins and observe them in
vivo. Other groups contain photoaffinity labels that could
be used for covalently cross-linking proteins to one another.
This allows scientists to see what the proteins interact with
in living cellseven weak interactions that are difficult
to detect by current methods.
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.
While inserting novel amino acids inside proteins is nothing
new, in the past such modifications had to be carried out
in the test tube, with the scientist doing all the manipulations
by hand. Now, the 21-amino acid bacterium uses its own "hands"
to make the modified proteins.
The Basis of the Technology
Schultz and his colleagues succeeded in making the 21-amino
acid bacteria by exploiting the redundancy of the genetic
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
(UAG, ACG, UTC, etc.). With only 20 amino acids used by the
organisms, not all of the codons are theoretically necessary.
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, called the amber stop codon, UAG, played
an important role in Schultz's research.
Schultz knew that if he could provide his cells with what
is known as an amber suppressora tRNA molecule that
recognizes UAGand also with an enzyme that loaded the
amber suppressor tRNA with an unusual amino acid, then he
would have a way to site-specifically insert the unusual amino
acid into any protein he wanted.
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. They just needed to add the novel amino acid to
the culture and grow the cells.
Using this method, Schultz and his colleagues last year
incorporated the unusual amino acid O-methyl-L-tyrosine into
proteins with fidelity greater than 99 percent, which is close
to the translation fidelity of natural amino acids. They have
since demonstrated the ability to incorporate several other
unusual amino acids into proteins, including the unusual amino
acid p-aminophenylalanine, which is described in the latest
Now, by adding "plasmids"circular, self-contained
pieces of DNA that express the metabolic genes necessary for
making p-aminophenylalaninethey have given the bacteria
the ability to synthesize their own unusual amino acids and
insert them into any protein coded for by an mRNA containing
a UAG codon.
With a fully autonomous 21 amino acid bacterium, they can
also compare this unique form of life to an analogous bacterium
that uses only the 20 natural amino acids and see how their
evolutionary fitness and survivability compare.
The article, "Generation of a 21 Amino Acid Bacterium" was
authored by Ryan A. Mehl, J. Christopher Anderson, Stephen
W. Santoro, Lei Wang, Andrew B. Martin, David S. King, David
M. Horn, and Peter G. Schultz and appeared in the ASAP online
edition of the Journal of the American Chemical Society
on January 4, 2003. See: http://pubs.acs.org/cgi-bin/asap.cgi/jacsat/asap/abs/ja0284153.html.
The article will appear in print later this year.
This work was supported by the U.S. Department of Energy,
through a National Science Foundation predoctoral fellowship,
and through a Jane Coffin Childs Memorial Fund for Medical