Making protein takes energy. A lot of it. And your cells are paying for it before they even get to the ribosomes! The charging starts with amino acid activation & tRNA charging, which is done by aminoacyl-tRNA synthetases (aaRS’s), which have the monumental task of recognizing the right amino acid & the right tRNA and getting them to join together. It takes a molecule of ATP, which gets split to AMP, “bypassing” ADP, so you have to spend another ATP to regenerate ADP to regenerate ATP. So it’s almost like it cost you 2 ATP just for the charging! (and then you’ll spend a GTP to make sure the right tRNA gets brought and another one to get things to scootch over in the ribosome… so 3 triphosphates (kinda 4) per peptide added). But the upfront charge is worth it because the ribosome needs to add the right thing! And when you actually take the charged tRNA to the ribosome, all the ribosome cares about is that the anticodon on the tRNA matches the codon on the mRNA with the protein’s instructions. So what’s the deal with these aaRS’s?

A little old text, then I’m mostly going to talk it out – and link to some good reviews & articles

and if you want more on translation – including a description in wedding terms… 

In the protein-making process of translation, amino acids are taken to the ribosome by transfer RNAs (tRNAs). Each tRNA will only accept one type of amino acid (but there are lots of identical copies of all of these things, so there’s hopefully plenty to go around). Each amino acid has at least one tRNA just for itself and it’s important that the right amino acids get on the right one because the ribosome won’t be able to tell what amino acid’s on it, just what tRNA it is (by “reading” a 3 RNA letter word on the tRNA called an anticodon that complements a codon on the mRNA. more on this genetic code:

It’s really important that there’s no identity theft allowed! Your cells have to pay for this added layer of security in the form of energy money with the help of a molecule called an aminoacyl tRNA synthetase (aka tRNA-ligase). In amino acid activation, an AMP molecule is added onto the amino acid from ATP. Then, in the tRNA charging step, the amino acid binds to the matching tRNA (the one whose anticodon matches the codon in the mRNA). The AMP is released in the process, leaving you with aminoacylated tRNA (aa-tRNA). 

We call it “charging” because phosphate bonds are “high energy” – they don’t like being held in one spot – especially next to other phosphate groups, which repel them – literally – because of the negative charge-negative charge situation. ATP’s happy to give up phosphate(s) because it’s like releasing a spring. more on this here: 

Why spend the money? Specificity! Ribosomes only make about 1 mistake every 10,000 amino acids. They can achieve this great accuracy because they use a strict verification process starting by making sure the right amino acid binds the right tRNA (and later a second layer of security makes sure the right tRNA binds the right codon) 

How does the aminoacyl synthetase select the right amino acid to attach to the matching tRNA? The correct one is favored because it has the highest affinity for the synthetase’s active site. That works fine for telling apart big things and little things (like glycine & tryptophan), but can mess up when things look similar (like valine & isoleucine) so you need a second step to check via hydrolytic editing. When tRNA binds the synthetase, it pushes the amino acid into a second proofreading pocket on the synthetase that does a closer check – the right one can’t fit into this pocket, so it stays safe but wrong ones can get in, so they get hydrolyzed & released. (note that some synthetases use different ways or don’t edit at all – more on this in the review article linked to at the end)

Then it has to make sure that the right tRNA gets attached to that activated amino acid, so it has to be able to recognize that correct  tRNA. There are different ways of doing this. Most match the anticodon (have 3 nucleotide-binding pockets shaped perfectly for the 3 RNA letters of the anticodon) – some instead or in addition recognize sequences in the acceptor stem.

So, now you’ve “paid” your deposit and ensured specificity of tRNA-amino acid pair, it’s time to start heading to the ribosome. But you’ll want to take a “security guard” to make sure you don’t go to the wrong place. Elongation factors (EF-G in bacteria and EF-1 in humans) travel with the aa-tRNA and don’t leave until they make sure that the codon and anticodon match (note that it’s the anticodon that matters here, not the actual amino acid, which is why that precaution during the charging step is important) – you don’t want to drop off imposters!

Once the EF sees all’s good, it “pays for the aa-tRNA’s parking” by hydrolyzing (using water to split) GTP into GDP (same concept as usage of ATP for energy, just a different nucleobase), & this time the energy’s being used for shape-shifting. When it’s bound to GTP the EF has one shape, but when it’s bound to GDP it has another shape. So, after splitting the GTP, it “splits” (falls off)

The EF drops off the incoming tRNA in the ribosome’s “A” spot (1 of 3 parking spots that fit in the ribosome at a time). The ribosome then carries out its enzymely functions, uniting the new amino acid with the growing peptide chain through a peptide bond union. The growing strand gets transferred to the new tRNA, so you have an awkward transition stage where the peptide’s still mostly in the P spot but it’s attached to a tRNA that’s in the A spot. Another elongation factor, (EF-Tu in bacteria or EF-2 in humans) comes in and moves things along, spending GTP to push the old tRNA (still attached to its codon) into the E spot (where it falls off), the tRNA with the growing chain into the P spot, and bringing a new codon into the A spot for the next amino acid to be added.

In a little more detail/jargon, there are 3 steps of each addition in the elongation phase:

  • tRNA selection (decoding)
    • aminoacyl-tRNA w/proper anticodon to match mRNA codon in A site
      • delivered by GTPase eEF1A (EFTu) in ternary complex w/GTP
      • eEF1A activated → hydrolyses GTP to enable tRNA to be fully accommodated into A site
  • peptide-bond formation
    • amino group of incoming amino acid attacks ester linkage on peptidyl-tRNA in ribosomal P site → growing chain transferred to tRNA in A site
    • as bond forms, ribosomal subunits adopt an “awkward” hybrid state – the acceptor ends shift over while the anticodon ends are stuck in place (the acceptor ends have more “wiggle room” but the anticodons are tightly bound to their matching codon (which is good cuz you don’t want them to slip)
  • translocation of mRNA-tRNA complex
    • another GTPase, eEF2 (EFG) recognizes the “awkward” hybrid state – spends GTP to translocate the mRNA-tRNA complex relative to the ribosome

Something special happens when a “stop codon” shows up in the A spot. This signals the end of the chain. Instead of a tRNA binding it, a protein termination factor binds and cleaves the chain off. more on translational termination:

The newly-made polypeptide can then make itself comfortable by folding into the optimal conformation so that the amino acids that like each other are kept together, ones that hate each other are kept apart, etc. like we talked about here: 

Read more:

A really great review article on aaRS’s: 

Rubio Gomez, M. A., & Ibba, M. (2020). Aminoacyl-tRNA synthetases. RNA (New York, N.Y.), 26(8), 910–936.

a review article on aaRS’s as drug targets (mostly anti-microbe & anticancer therapeutics) 

Kwon, N.H., Fox, P.L. & Kim, S. Aminoacyl-tRNA synthetases as therapeutic targets. Nat Rev Drug Discov 18, 629–650 (2019). 

articles about artificial aaRS’s that are actually ribozymes called flexizymes:

Murakami, H., Ohta, A., Ashigai, H. et al. A highly flexible tRNA acylation method for non-natural polypeptide synthesis. Nat Methods 3, 357–359 (2006). 

Goto, Y., Katoh, T. & Suga, H. Flexizymes for genetic code reprogramming. Nat Protoc 6, 779–790 (2011). 

An article on energy efficiency problems in in vitro translation systems:

Szaflarski, W., Nierhaus, K.H. Question 7: Optimized Energy Consumption for Protein Synthesis. Orig Life Evol Biosph 37, 423–428 (2007). 

more on PURE systems: 

If you have access to it (might have to request through your library) I highly recommend this book chapter to anyone wanting to learn more about translation: The Ribosome and Protein Synthesis by Paul Huter, Michael Graf, and Daniel N. Wilson 

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