TSRI Scientists Create New Strain of Yeast with 21-Amino Acid Genetic Code

By Jason Socrates Bardi

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 amino acids.

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 organisms.

"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 acids—and only those 20—to make proteins.

The question is why did life stop with 20 and why these particular 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 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 protein–protein 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 cells—even 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 proteins—an 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 studies.

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 code.

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 codon—or 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 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, UAG, played an important role in Schultz's research.

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" pair—a 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 acid.

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. See: http://www.sciencemag.org.

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A team led by Professor Peter Schultz is reporting a general method for adding unnatural amino acids to the genetic code of a type of yeast. Photo by BioMedical Graphics.

Novel Amino Acids Added to the Genetic Code of S. Cerevisiae: