The development of new orthogonal aminoacyl-tRNA synthetase/tRNA pairs has led to the addition of approximately 70 unnatural amino acids (UAAs) to the genetic codes of Escherichia coli, yeast, and mammalian cells. These UAAs represent a wide range of structures and functions not found in the canonical 20 amino acids and thus provide new opportunities to generate proteins with enhanced or novel properties and probes of protein structure and function.

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Adding New Chemistries to the Genetic Code

Although of limited metabolic diversity, methanogenic archaea or methanogens possess great phylogenetic and ecological diversity. Only three types of methanogenic pathways are known: CO(2)-reduction, methyl-group reduction, and the aceticlastic reaction. Cultured methanogens are grouped into five orders based upon their phylogeny and phenotypic properties. In addition, uncultured methanogens that may represent new orders are present in many environments. The ecology of methanogens highlights their complex interactions with other anaerobes and the physical and chemical factors controlling their function.

Metabolic, phylogenetic, and ecological diversity of the methanogenic archaea.

http://hoffman.cm.utexas.edu/courses/wood_lungdahl_1.pdf

Acetogenesis and the Wood-Ljungdahl Pathway of CO2 Fixation

We describe a general and rapid route for the addition of unnatural amino acids to the genetic code of Saccharomyces cerevisiae. Five amino acids have been incorporated into proteins efficiently and with high fidelity in response to the nonsense codon TAG. The side chains of these amino acids contain a keto group, which can be uniquely modified in vitro and in vivo with a wide range of chemical probes and reagents; a heavy atom-containing amino acid for structural studies; and photocrosslinkers for cellular studies of protein interactions. This methodology not only removes the constraints imposed by the genetic code on our ability to manipulate protein structure and function in yeast, it provides a gateway to the systematic expansion of the genetic codes of multicellular eukaryotes.

http://science.sciencemag.org/content/sci/301/5635/964.full.pdf

An Expanded Eukaryotic Genetic Code

Although chemists can synthesize virtually any small organic molecule, our ability to rationally manipulate the structures of proteins is quite limited, despite their involvement in virtually every life process. For most proteins, modifications are largely restricted to substitutions among the common 20 amino acids. Herein we describe recent advances that make it possible to add new building blocks to the genetic codes of both prokaryotic and eukaryotic organisms. Over 30 novel amino acids have been genetically encoded in response to unique triplet and quadruplet codons including fluorescent, photoreactive, and redox-active amino acids, glycosylated amino acids, and amino acids with keto, azido, acetylenic, and heavy-atom-containing side chains. By removing the limitations imposed by the existing 20 amino acid code, it should be possible to generate proteins and perhaps entire organisms with new or enhanced properties.

Expanding the genetic code.

Pyrrolysine is a lysine derivative encoded by the UAG codon in methylamine methyltransferase genes of Methanosarcina barkeri. Near a methyltransferase gene cluster is thepylT gene, which encodes an unusual transfer RNA (tRNA) with a CUA anticodon. The adjacent pylS gene encodes a class II aminoacyl-tRNA synthetase that charges the pylT-derived tRNA with lysine but is not closely related to known lysyl-tRNA synthetases. Homologs of pylS and pylT are found in a Gram-positive bacterium. Charging a tRNACUA with lysine is a likely first step in translating UAG amber codons as pyrrolysine in certain methanogens. Our results indicate that pyrrolysine is the 22nd genetically encoded natural amino acid.

https://science.sciencemag.org/content/sci/296/5572/1459.full.pdf

Pyrrolysine encoded by UAG in Archaea: charging of a UAG-decoding specialized tRNA.

Genes encoding methanogenic methylamine methyltransferases all contain an in-frame amber (UAG) codon that is read through during translation. We have identified the UAG-encoded residue in a 1.55 angstrom resolution structure of the Methanosarcina barkeri monomethylamine methyltransferase (MtmB). This structure reveals a homohexamer comprised of individual subunits with a TIM barrel fold. The electron density for the UAG-encoded residue is distinct from any of the 21 natural amino acids. Instead it appears consistent with a lysine in amide-linkage to (4R,5R)-4-substituted-pyrroline-5-carboxylate. We suggest that this amino acid be named l-pyrrolysine.

https://science.sciencemag.org/content/sci/296/5572/1462.full.pdf

Carey M. James

Papers

Pyrrolysine encoded by UAG in Archaea: charging of a UAG-decoding specialized tRNA.

A new UAG-encoded residue in the structure of a methanogen methyltransferase.

The amber codon in the gene encoding the monomethylamine methyltransferase isolated from Methanosarcina barkeri is translated as a sense codon.