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Codon Optimization & Codon Usage Bias

What color is the sky before a storm? Brits may write “grEy” while Americans write “grAy” but both recognize it as meaning a color between white & black. Similarly, the 3-letter genetic “words” that “spell” the amino acid building blocks of proteins can be written with different spellings. All organisms can understand these words (the GENETIC CODE is universal), BUT different organisms prefer different spellings (CODON BIAS). If you want to get an organism to express a protein for you, you might want to “be polite” and use their spelling! This is one aspect of CODON OPTIMIZATION (which also includes considering the codon in context, making sure you don’t cause the DNA or RNA to fold up weird, and that you don’t accidentally introduce sequences the cell thinks are ribosome binding sites or something) full text & figures: http://bit.ly/codonbias Different amino acids are specified by 3-letter RNA “words” called CODONS. There are 4 nucleotide letters – A, C, G, & T/U – so 64 possible codons. BUT there are only 20 (common) amino acids. 3 of the codons don’t spell an amino acid – instead they spell STOP & signal the end of the protein. But that still leaves 61. So some amino acids have multiple codons (we call this DEGENERACY or redundancy). BUT any 1 codon will only ever spell 1 amino acid (NOT ambiguous) if you want to learn more about how this was discovered: http://bit.ly/2old6h6 One part of tRNA binds a specific amino acid and the other end contains a 3-nucleotide ANTICODON that is complementary to the matching 3-letter CODON on the mRNA. Different tRNAs have different ANTICODONS & carry different amino acids. Because of degeneracy, multiple servants may bring the same amino acids (e.g. “gray” and “grey” both bring the same colored charm), but the ribosome’s a bit of a snob – it will only add that amino acid if it’s brought by the right servant. And the servant it wants is determined by the CODON in the mRNA. Each tRNA only ever brings 1 type of amino acid, BUT some tRNA can read multiple codons. To make the making more likely, we can introduce “silent mutations” for “codon optimization” - Organisms make each type of tRNA servant, but how many of each they have depends on how popular the corresponding codon spelling is – they stock up on the ones they have to use the most. You can think of it kinda like one of those magnet poetry sets - if you’re selling a color-themed word magnet set in America, you’d probably include more “grAy” than “grEy” because that’s what your customers will demand more of. And vice versa in England. If an American needs a “grEy” magnet, this might slow down their poem-making because they’ll have to do a bunch of digging through magnets to find one. They might even “give up” Similarly, when a ribosome’s traveling along and it comes to a “rare codon” it’ll have to stop and wait for the matching tRNA. If they have to wait too long, “translational stalling” can lead to things like premature termination (“giving up” before the full protein’s made) or mistake-making (things like skipping letters or sticking in the wrong ones). However, sometimes a bit of a brief breather can be a benefit - cells use it to the forming protein’s advantage because “pausing” can allow for proper folding of the part of the protein that’s been made so far. Another time rareness can be useful for cells is as a way to control gene expression. If an mRNA contains a lot of rare codons, the corresponding protein will likely be translated more slowly, so less of it will be made. If you want to sell a magnet set, consider your audience’s preferences – replace those “grAy” magnets with “grEy” or vice vera. Similarly, we can order genes codon-optimized for the cell type we want to express them in (e.g. bacteria, yeast, insect cells). Companies like GenScript use algorithms to determine what the optimal codons are based in large part on what codons are most “popular” in those cells. Then they synthesize the genes to match. For example, there are 6 codons that spell Leucine (Leu, L) & E. coli have 4 Leu tRNAs. The tRNA that recognizes CUG is very abundant, whereas the one for CUA is rare, so swapping CUA for CUG can lead to the recruitment of a more common servant and less holdup. I said “in large part” based on relative #s, because it’s not just quantity that matters - even when a tRNA can read 2 codons, 1 form is usually “preferred” – the tRNA can read either, but it works better for one of them. So even if the cells have lots of the tRNA that can read that codon, you’ll get better results if you use the one it prefers. Going back to our E. coli codons: The phenylalanine (Phe, F) codons UUU & UUC are read by the same tRNA, but that tRNA has a 3’-AAG-5’ anticodon, so it prefers UUC, which it can bind perfectly to. So if you change a sequence from UUU to UUC you might have better luck even though they use the same codon. GenScript webinar    • Codon optimization: Why & how to desi...