The Skaggs Institute
for Chemical Biology 2004

Scientific Report 2004

Expanding the Genetic Code

P.G. Schultz, E. Brustad, S. Chen, J. Grbic, J. Melnick, J. Mills, K.H. Min, M. Mukherji, A. Nagle, S. Schiller, E. Tippmann, J. Turner, J. Wang, J. Xie, L. Zheng

Almost all processes of living cells, from gene regulation and information processing to photosynthesis, are carried out by proteins. The 20 amino acids used as building blocks in the synthesis of proteins are connected in different combinations to give polymeric structures consisting of anywhere from tens to thousands of amino acids. What is amazing is that every form of life on Earth uses the same set of 20 amino acids to make all proteins. Indeed, this set of amino acids is the basis for the genetic code, the code that specifies the relationship between the nucleotide sequence of a gene and the amino acid sequence of a protein. This fact leads to the rather interesting question of why every form of life has the same set of building blocks. Why not 21 or more? Moreover, if we can add new amino acid building blocks to the genetic code, will we be able to create proteins or even new organisms with enhanced chemical, physical, or biological properties?

We have begun to address this question by using a number of chemical and molecular biological methods to add new components to the protein biosynthetic machinery of bacteria. This machinery consists of the ribosome, which binds mRNA (a short-lived single-stranded copy of the DNA that encodes a protein) and translates it into a protein sequence. The translation is accomplished by an adapter molecule called tRNA. The genetic code is enforced by enzymes (tRNA aminoacyl synthetases) that load each tRNA with 1 of the common 20 amino acids specified by the genetic code. By adding new components to this biosynthetic machine, we showed that we can effectively expand the genetic code of Escherichia coli, yeast, and mammalian cells by adding new amino acids including photoaffinity labels, keto amino acids, heavy atom–containing amino acids, and amino acids with altered electronic and steric properties. In addition, we made the first synthetic autonomous organism with a 21 amino acid genetic code and are exploring its ability to evolve in response to a variety of environmental stresses. In the past year, we extended this method to mammalian cells; encoded unnatural amino acids with 4-base frame-shift codons; incorporated redox-active, photocaged, and isotopically labeled amino acids; and created a new orthogonal tRNA-synthetase pair in yeast.

Our goals for 2005 are to incorporate unnatural amino acids into multicellular organisms; show that amino acids with altered backbones can be incorporated; incorporate metal-binding amino acids, fluorescent amino acids, and posttranslationally modified amino acids; and determine x-ray crystal structures of mutant synthetases that encode heavy-atom, keto, and glycosylated amino acids.

Publications

Anderson, J.C., Wu, N., Santoro, S.W., Lakshman, V., King, D.S., Schultz, P.G. An expanded genetic code with a functional quadruplet codon. Proc. Natl. Acad. Sci. U. S. A. 101:7566, 2004.

Tian, F., Tsao, M.L., Schultz, P.G. A phage display system with unnatural amino acids. J. Am. Chem. Soc. 126:15962, 2004.

Xie, J., Wang, L., Wu, N., Brock, A., Spraggon, G., Schultz, P.G. The site-specific incorporation of p-iodo-L-phenylalanine for structure determination. Nat. Biotechnol. 22:1297, 2004.

Zhang, Z., Alfonta, L., Tian, F., Bursulaya, B., Uryu, S., King, D.S., Schultz, P.G. Selective incorporation of 5-hyroxytryptophan into proteins in mammalian cells. Proc. Natl. Acad. Sci. U. S. A. 101:8882, 2004.

Zhang, Z., Gildersleeve, J., Yang, Y.Y., Xu, R., Loo, J., Uryu, S., Wong, C.-H., Schultz, P.G. A new strategy for the synthesis of glycoprotein. Science 303:371, 2004.