Expanding the Genetic Code: Discovery of Two New Amino Acids

In high school biology classes, we learn that there are 20 amino acids. But in the past two decades, researchers have discovered two addi­tional amino acids that are incorporated into natural genetic codes – selenocysteine (Sec) and pyrrolysine (Pyl). Every amino acid has its own transfer RNA (tRNA) and aminoacyl-tRNA synthetase (aaRS). The tRNA molecule binds specifically to the amino acid and brings it to the site of protein synthesis. The enzyme aminoacyl-tRNA synthetase specifically attaches the amino acid to its tRNA.

The twenty-first amino acid, Sec, has its own specific codon and tRNA, but it lacks its own aaRS. In fact, Sec is synthesized through the modification of the canonical amino acid serine after its attach­ment to the Sec tRNA. Pyl, on the other hand, has its own specific codon, tRNA, and aaRS. Thus, the entrance of Pyl into the natural genetic codes of certain organisms also marks the evolution of the first non-canonical aaRS and tRNA.

The importance of Pyl in protein evolution is one reason why this amino acid interests Dieter Söll, Sterling Professor of Molecular Biophysics & Biochemistry. Research done on Pyl in the Söll laboratory has helped to better understand the evolution of proteins and of the genetic code, which has practical applications in biotechnology. “What if you expand the genetic code by adding another amino acid? That’s what happened with Pyl,” said Patrick O’Donoghue, a postdoctoral fellow in Professor Söll’s lab. “The same idea is used in biotechnology. You want to incorporate new amino acids in the genetic code – fluorescent or crosslinking amino acids, for example.”

Pyl evolved quite early, before the last universal common ancestor on Earth. But today, Pyl is very rare. It is found in only seven organisms, and in each of those organisms it is found in only a few genes. Of those seven organisms, two are bacterial and five archaeal, and all live off of methylamine, the compound that gives fish its unique smell. “What we find interesting is that you would create a whole new amino acid, and then maintain a whole new coding method to live off of stinky fish,” said O’Donoghue. In a recently published paper in Nature, Söll and colleagues study the Pyl tRNA synthetase (PylRS) and tRNA (tRNAPyl) from the bacterium Desulfitobacterium hafniense. Through in vitro and in vivo studies, they demonstrated that the PylRS-tRNAPyl pair was “orthogonal.” In other words, the PylRS and tRNAPyl interact specifically with one another and not with the aaRS or tRNA of any other endogenous amino acids.

“In a pair that is not orthogonal, two things could happen – the synthetase could interact with other tRNAs, or the tRNA could interact with other tRNA synthetases,” said O’Donoghue. “But the Pyl system is orthogonal, so it does not inter­fere with normal protein synthesis.” The structure of the PylRS-tRNAPyl pair gives a molecular explanation for its orthogonality. “The pyrrolysine tRNA synthetase is completely different from all other synthetases,” said Söll. This difference arises from unique appendages that allow PylRS to interact specifically with tRNAPyl. PylRS also includes a domain that is conserved among many aaRS proteins, but this similarity only holds on a very general scale. The conserved parts of the aaRS protein are in fact modified to make specific contacts with tRNAPyl.

A better understanding of the molecular struc­ture of the PylRS and tRNAPyl opens up the possibility of engineering the genetic code. “The orthogonality of the aaRS-tRNA pair makes the Pyl system a good tool for incorporating other amino acids in the genetic code,” said Sharath Gundllapalli, another postdoctoral fellow in the Söll lab. Using the Pyl system, researchers can specifically encode the modification of one non-canonical amino acid in a protein and then deter­mine the role of that amino acid. Non-canonical amino acids created by post-translational modifi­cation are included in proteins involved in many fundamental processes in the cell. Understanding their roles is important in understanding what goes wrong with these processes in human disease and could eventually have implications for drug development and design.

Possibly as far back as 3 billion years ago, Pyl evolved its own tRNA and aaRS, expanding the number of amino acids to 22. The molecular basis of orthogonality in the aaRS-tRNA pair reveals how the addition of Pyl to the genetic code took place. This orthogonality was crucial to ensuring the reliability of protein synthesis. Because of their research on PylRS and tRNAPyl, researchers in Söll lab now have a better understanding of the evolution and diversity in the genetic code.

Further Reading

• Blackman, Stuart. “Discovering the 21^st Amino Acid … Again? The Scientist. 11 Oct 2004. 18(19):22. Feb 2009. https://www.the-scientist.com/article/display/14979.
• Nozawa, K. et al. Pyrrolysyl-tRNA synthetase-tRNA(Pyl) structure reveals the molecular basis of orthogonality. Nature, 2009. 457:1163-1167.
• Yarnell, Amanda. “22^nd Amino Acid Identified.” Chemical & Engineering News. 27 May 2002. American Chemical Society. 80(21):13. Feb 2009. https://pubs.acs.org/cen/topstory/8021/8021notw1.html.