Expanding the Genetic Code:
TSRI Scientists Synthesize 21-Amino Acid Bacterium
By Jason Socrates Bardi
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 proteins—instead
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," he says.
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 molecular
medicines.
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 acids—and only those 20—to
make proteins.
"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 pyrrolysine—slightly 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 cells—even 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 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 codon—or 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 codons—they
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 suppressor—a tRNA molecule that recognizes UAG—and
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 report.
Now, by adding "plasmids"—circular, self-contained pieces of DNA
that express the metabolic genes necessary for making p-aminophenylalanine—they
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 Research fellowship.
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