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.



"We have effectively removed a billion-year constraint on our ability to manipulate the structure and function of proteins," says TSRI investigator Peter Schultz.