Unnatural/non-canonical amino acid incorporation by "genetic code expansion" inspired by pyrrolysine

Pyrrolysine is the 22th genetically-encoded proteinogenic amino acid (protein letter). It was discovered in 2002 & it’s only found in a few bacteria and archaea, who use it to help break down carbon-y things into methane (which humans can potentially use as an alternative energy source). And they’re able to use this special amino acid by rewriting their genetic code slightly so that this special protein letter replaces one of the protein-making stop signs, UAG (there were 3 already, so they won’t miss one!). This lets them introduce this “unusual” 22nd amino acid into their proteins (and scientists are using this “orthogonal” strategy to introduce unusual amino acids into other proteins in the lab). ⠀ ⠀ blog form (refreshed from last December): http://bit.ly/pyrrolysine ⠀ ⠀⠀ Protein letters (amino acids) are spelled as 3-letter RNA letter (nucleotide) words called codons (e.g. CAG “spells” the amino acid glutamine, whereas CGG spells arginine). Another type of RNA, transfer RNA (tRNA), “charged” with the corresponding protein letter (i.e. it has an amino acid stuck to it) brings that letter to the ribosome (with the help of a protein called an elongation factor) when a codon complementing the tRNA’s 3-letter anticodon shows up in the ribosome’s “entry-way.” The ribosome keeps on doing this, adding one amino acid letter for each 3-RNA letter step (codons are read non-overlappingly) until it reaches a stop codon. Then, instead of a tRNA binding, a protein called a release factor, acts as a “fake tRNA” - it comes in and helps use water to break the completed peptide off of the tRNA holding it. These release factors don’t have direct complementary base pairing for super-stickiness (the release factor is a protein so it can only “pretend” to be like tRNA - plus it has to be able to bind to 3 different stop codons, so it couldn’t have one-to-one pairing even if it were legit RNA!) As a result, the release factors can be “beaten to it” by tricky tRNAs.⠀ ⠀ Yesterday we looked at how the amino acid selenocysteine (Sec) has a tRNA with an anticodon that complements the “stop codon” UGA, so it’s able to fool the ribosome into adding it when it reaches UGA. But this only happens really really rarely. Almost always, the ribosome stops when it gets to UGA - it’s only in a couple dozen mRNAs that Sec can sneak in - because those mRNAs have a special loopy part after the end of the protein-spelling part (in the 3’ untranslated region (UTR)) called the SECIS (selenocysteine insertion element). The SECIS binds a special Sec helper protein called SECIS-binding protein 2 (SBP2) which binds to charged tRNASec and its special elongation factor EFSec and interacts with the ribosome, keeping Sec at the ready to sneak in when the ribosome encounters UGA and stalls (it will only sneak in at UGA, not at other stop codons because the Sec-tRNA has an anticodon that complements UGA) http://bit.ly/selenocysteinetrickery ⠀ ⠀ A different mechanism is used to sneak pyrrolysine in - a less “sneaky” one - instead of having a Pyl being a “sometimes spelling”, it’s turned into an “almost always” spelling - the few organisms that use it have basically rewired themselves so that UAG is mostly just like any normal codon. Some Pyl-using organisms still uses UAG as a stop codon, but they compete. They tend to use UAG as stop really rarely and when it is meant to mean stop it’s usually followed by another nearby (nonambiguous) stop codon just in case. There’s some controversy about the potential role of a downstream enhancer sequence called PYLIS that was proposed to play a similar role to SECIS, but doesn’t seem as important. UAG is sometimes called an “amber” codon and, since tRNAPyl suppresses the stoppage of the ribosome at UAG, it’s called an “amber suppressor.”⠀ ⠀ That’s how Pyl gets into the protein, but how does Pyl itself get made? Some people argue that Pyl is actually the 21st amino acid - that Sec doesn’t count because, although the modification happens before adding the amino acid to the protein, it happens after it’s loaded onto its tRNA carrier - Sec-tRNA is first charged with serine, and then, through a couple steps, Ser’s -OH gets swapped out for Sec’s -SeH. In contrast, Pyl is made before it gets loaded onto its corresponding tRNA⠀ ⠀ A few years ago, scientists made significant progress in figuring out how this happens, proposing a reaction mechanism that involves 3 key enzymes, PylB, PylC, & PylD. https://www.nature.com/articles/natur... ⠀ much more & links to key papers & reviews in the blog! http://bit.ly/pyrrolysine