If your ribosome stalls, who you gonna call? Ribosome Associated Quality Control (RQC)! When it comes to making functional proteins, amino acid identity theft isn’t the only thing you have to worry about! Problems during translation (protein-making) can cause a “paper jam” in the protein-making machinery, so your cells have mechanisms to “open up” that machinery and remove & “shred” the partially-made products. Let me have a go at explaining RQC and NO-GO mRNA processing!⠀

Proteins are made up of long chains of “letters” called amino acids which have a generic backbone part and 1 of 20 unique side chains (aka “R groups”) which sticks out like a charm from a charm bracelet and affects how the chain of amino acids (polypeptide) folds up into a functional protein. TRANSLATION is the process by which proteins are pieced together one amino acid letter at a time in the order dictated by messenger RNA (mRNA) instructions. I liken it to a “wedding” of amino acids. The unions are called peptide bonds and these backbone-to-backbone linkages are carried out in a ribosomal “drive-thru chapel,” with amino acids binding to “reserved parking spots” on an mRNA “road”

It can be easier to think of it in these abstract terms because ribosomes themselves are pretty biochemically complicated… In less abstract terms, the ribosome is a big complex made up of 2 smaller big complexes, each composed of lots of ribosomal RNAs (rRNAs) and proteins. The 2 “halves” are the large ribosomal subunit (60S in humans), which is the bulk of the chapel, and the large ribosomal subunit (40S in humans) that holds the mRNA “road”. It serves double-duty – it provides an altar, holding the mRNA in place, exposing the 3-nucleotide-letter codons that “spell” 1 amino acid letter, and it acts as the “priest,” mediating the peptide bond formation.⠀

Amino acids get rides to this chapel from transfer RNAs (tRNAs). On one end these tRNAs attach to the amino acid and at the other end they have a 3-letter “anticodon” that complements the codon that spells the amino acid it’s attached to. It serves as a sort of “license plate” in a couple senses of the term -> it provides a unique identifier (there are 20 (common) amino acids and each has at least 1 tRNA dedicated just to it). And it “licenses” binding to the matching codon.⠀

Only when that codon shows up in the ribosomes “A” site does the corresponding tRNA have “permission” to park there (an elongation factor “security guard” spends energy money to ensure this. And to make sure there’s no amino acid “identity theft” going on (the ribosome can’t tell who’s in the tRNA limo, just what limo it is), there are mechanisms (requiring more energy money) for amino acid “passenger” to get in the right limo. ⠀

Throughout the translation process, as new tRNA limos come in, the growing peptide chain is handed over to them, then another elongation factor helps the whole thing shift over so a new codon is in the A site. There are only 3 sites: A where they come in, P where the growing chain is held, and E where the passenger-less limos fall off the road from so they can be reused.⠀

As a peptide’s being made, it gets threaded through a tunnel. Think of it as traveling up the chapel’s bell-tower/chimney. So it’s hidden for a while before it emerges into the “light” of the cytoplasm (general cellular interior), where it starts to fold into its 3-D structure & where other proteins can find it – and potentially bind it. Sometimes, the emerging end enters a chaperone protein that acts as a sort of “waiting room” that helps ensure proper folding by sheltering parts that come out before the parts that they need to interact with in the folded form.⠀

If the wedding goes according to plan, a “stop codon” will show up in the A site and cause the binding of release factor “bouncers” that kick everything out so the chapel can be recycled for another wedding.⠀

But if the ribosome stops and there’s not a stop codon there, no “bouncer” comes, and the ribosome’s stuck with a partially-made protein (attached to the tRNA). This can happen if there are problems like steep hills in the road (strong secondary structure in the mRNA), potholes (mRNA damage), not enough of the limo you need (can happen for rarely-used codons for which cells don’t stock up on their limos cuz it’d be a waste of time). And it causes the ribosomal machinery to stall. Multiple ribosomes are usually translating the same mRNA at the same time (polyribosomes) (share the road!), so another ribosome can crash into it & trigger a “call to AAA” – initiate the no-go decay pathway (and speaking of “AAA,” a AAA+ ATP-ase is gonna help save the day later by extracting the peptide chain…) more here: http://bit.ly/2KWfcPo⠀

Your cells deal with it kinda like you’d deal with a paper jam in your printer. They split apart the ribosome into its 2 “halves” – the large subunit and the small subunit – pulling out the stuck stuff (the tRNAs, peptide chain, & mRNA), checking the parts to make sure all’s ok so it won’t happen again, and breaking down & recycling parts from the useless stuff (the partial peptide chain that’s like the crumpled-up inky paper that was jammed). This is carried out by a ribosome-associated quality control complex (RQC).⠀

Let’s start with the partial protein (peptide) – degrading the peptide is important because partial proteins can be toxic to the cell – for example, they might have the part of a protein that binds something but not the part that does something to the thing it binds. So it hogs the thing without doing a thing to the thing. Also, partial proteins don’t have a chance to fold properly, so they can clump together (aggregate) and cause problems.  ⠀

So the proteasome is the way for them – the proteasome is a protein recycling complex which cuts up proteins into pieces so that the amino acid letters can be reused and/or salvaged for smaller parts. But to get a “ticket” to the proteasome, a protein needs to be ubiqutinated. Ubiquitin (Ub) is a small protein – chains of which can get attached to proteins by ubiquitin ligases. The peptide chain to be extracted in ribosome-associated quality control (RQC) gets tagged through ubiquitination by a Ub ligase called Listerin (no “e”, so it’s not like the mouthwash :P). ⠀

Such tagging is made easier by splitting apart the stalled ribosome to give the ligase access to the protein – especially the protein’s lysine amino acids (Ubiquitin links to other proteins through lysine residues (lysine’s one of the amino acids)). When the ribosome gets split into the 2 halves, the peptide is still stuck to the tRNA stuck to the 60S subunit, with the peptide stuck on its climb out the chimney. ⠀

The splitting reveals binding sites for RQC factors including that ubiquitin ligase. But it can only do its tagging job if there are accessible lysines. Depending on where the stalling occurs, various lengths of the peptide chain might be sticking out of the chimney. If lysine residues have emerged from the chimney and are within “reaching distance” of the ribosome-bound ligase, the peptide can get ubiquitinated. But if there aren’t but there are lysine residues still in the chimney, “CAT” tailing offers a way to push them out -> an enzyme adds alanine and threonine amino acids to the end and those lysine’s get pushed out the chimney and can get ubiquitinated. I got to learn about that at a meeting a couple years ago from Jonathan Weissman. Awesome stuff! more about CAT-tailing (official form – Weissman’s paper): https://bit.ly/306wn6L  And another group found that even proteins without Lys’s pushed out could benefit from CAT-tailing because the CAT tail itself can act as a degron (degradation signal). https://go.nature.com/2Mw2ODD

So that takes care of the partial protein, which you definitely want to get rid of. But what about the tRNA? They might be perfectly usable, but you want to make sure the limos are ok before reselling them to be used to take new bride/grooms. The proteasome’s made for proteins (hence the name), so you’ve got to separate the tRNA from the peptide before you send the peptide off.⠀

There’s been some controversy about how this is done. But at that same meeting a couple years ago I learned of some exciting research from Susan Shao & her lab at Harvard that sheds insight into this process. ⠀

There’s another time tRNA & peptide chain have to be separated – when the chain is finished (a stop codon recruits a termination factor protein (eRF-1 in humans) that hydrolytically cleaves the peptide-tRNA bond.⠀

But something different happens in this stalled-ribosome case. The peptide is tagged for degradation, but the tRNA isn’t because it might still be usable – but you want to check. If you were to just separate the 2 the tRNA wouldn’t have any evidence of having been involved in any problems. But what if the tRNA had some problem that contributed to the problem in the first place? You don’t want to just stick it back into circulation.⠀

So it gets “tagged” by removing its end. All tRNA has the same end – a “CCA” adaptor that allows it to bind to an amino acid. After a crash, the peptide chain gets removed by cutting the tRNA, NOT the peptide-tRNA bond. And, since tRNA is a a nucleic acid, you need a nuclease. Nucleases are like DNA/RNA scissors. Some chew from the ends (exonuclease) while others cut in the middle (endonuclease). We need an endonuclease here, and the one that’s involved is named ANKZF1 (Vms1 in yeast)⠀

It cuts off that 3 nucleotide adapter. When it does so it leaves a mark in the form of a 2’-3’ cyclic phosphate. Which your cell thinks is weird – linking between RNA letters usually involves the 2’ “leg” so you’ve got to free it up if you want to add that adapter back on (which you have to do in order for it to get reused).⠀

Because all tRNAs have this same adapter, the same enzyme (TRNT1) can add it back once it gets doctor’s clearance. If there is a problem with the tRNA’s folding, TRNT1 will add a double-tag “2 CCA” which targets it for degradation.⠀

A couple years ago they were still searching for the enzyme(s) that do this part, but they were able to figure out the rest of the process because they purified all the different players they did know & tested what combinations did (and didn’t) do what. And they could use an enzyme from a bacteria-infecting virus (phage) to do that decyclizing of the phosphate. It’s some really beautiful biochemistry and you can read the original article here: https://go.nature.com/2IKV2WL⠀

and/or a summary here: https://go.nature.com/2Dy0hEY⠀

And last year they found it! ELAC1: https://bit.ly/2XxZezb 

This biochemical story reminds me of a different kind of story – this time when I was a kid and my shoe got stuck in an escalator. My foot slipped right out so it was fine, but they made me and my parents go through this whole process of taking pictures of my foot to have evidence it was fine in case we tried to sue them or something. Then they paid us like $50 (for my probably ~$15 Payless shoe) All was fine but they lost money. And they had to check the escalator too so it was out of service for like a week. Moral of the story (watch your shoes on escalators and, more importantly) this checking’s an involved, expensive (energy, time, & resource-wise) process, so you don’t want to do it all the time, just as needed.⠀

more about translation: http://bit.ly/proteintranslation

more about the ubiquitin-proteasome system (the less-well-known UPS!): https://bit.ly/ubiquitinylation 

more about all sorts of stuff: #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0 ⠀

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