Road traffic usually works because most people obey the laws. The laws don’t need to be stated on every occasion, because we have shortcuts to remember them: symbols in the form of signs, lane markers, and lights. People drive on the agreed-on side of the street (right in the U.S., left in the UK). They stop at red and go on green. An eight-sided red stop sign is a familiar indicator for drivers who don’t know English. When everyone obeys the laws, the choreography is stunning when seen from above in time-lapse.
Some who have driven abroad know the anxiety of unfamiliar customs: steering wheels on the wrong side of the car, different expectations about right of way, when or not to beep, and so forth. Fortunately, all eukaryotic cells use a universal set of signals, which work well unless they are disobeyed. Those include stop codes in DNA.
In Science, Michael R. Lawson and six colleagues play the role of forensic investigators, figuring out what goes wrong when mRNA transcripts run a red light, so to speak. Their paper, “Mechanisms that ensure speed and fidelity in eukaryotic translation termination,” begins with a statement of the law: “How Translation Stops.” It includes a shocking statistic:
Protein synthesis concludes when a ribosome encounters a stop codon in a transcript, which triggers the recruitment of highly conserved release factors to liberate the protein product. Lawson et al. used traditional biochemical methods and single-molecule fluorescence assays to track the interplay of release factors with ribosomes and reveal the molecular choreography of termination. They identified two distinct classes of effectors, small molecules and mRNA sequences, that directly inhibited the release factors and promoted stop codon readthrough. These findings may buttress ongoing efforts to treat diseases caused by premature stop codons, which cause 11% of all heritable human diseases. [Emphasis added.]
The diseases include “cystic fibrosis, muscular dystrophy, and hereditary cancers.” Correct translation termination is a vital process, therefore, occurring constantly in every cell; it “must occur rapidly and accurately.”
Since translation is a single-file process, a better analogy than road traffic might be a paper tape reader connected to a 3-D printer. It can read and translate any tape to build any part, but by convention, a particular set of dots means stop, cut, and eject. Then the reader lets in the next paper tape to translate. The incoming paper tapes in cells are the messenger-RNA (mRNA) transcripts from DNA in the nucleus. The readers and translators are ribosomes. The translated tapes are the polypeptides that will become proteins.
A stop codon (typically UAA, but sometimes UAG or UGA) is not translated; it summons additional molecular machines to release the polypeptide and start translating the next one. When the ribosome correctly reads the stop codon, here’s what happens:
Protein synthesis concludes when a translating ribosome encounters a stop codon at the end of an open reading frame, triggering recruitment of two factors to liberate the nascent polypeptide: eukaryotic release factor 1 (eRF1), a tRNA-shaped protein that decodes the stop codon in the ribosomal aminoacyl-tRNA site (A site) and cleaves the peptidyl-tRNA bond, and eukaryotic release factor 3 (eRF3), a GTPase that promotes eRF1 action. After translation termination, the ribosome, peptidyl-tRNA site (P site) tRNA, and mRNA are released by recycling.
Zooming in on the Process
These factors were known to play roles in termination, but the details were unclear. This team zoomed in on the process by labeling eRF1 and eRF3 molecules with green fluorescent dyes so that they could observe the details when the ribosome reads the stop codon:
We found that the two eukaryotic release factors bound together to recognize stop codons rapidly and elicit termination through a tightly regulated, multistep process that resembles transfer RNA selection during translation elongation. Because the release factors are conserved from yeast to humans, the molecular events that underlie yeast translation termination are likely broadly fundamental to eukaryotic protein synthesis.
They also found that binding of eRF1 is fast in the presence of eRF3, but slow without it. It needs to be fast; otherwise, inappropriate transfer RNAs (tRNA) might compete for occupancy of the A site. The interaction dynamics imply that adequate concentrations of eRF3 must be present at all times to prevent stop codon readthrough.
Further monitoring showed that the two factors bind together before entering the ribosome. “Thus,” they conclude, “eRF3 is a chaperone that delivers eRF1 to ribosomes halted at stop codons, and eRF3 departure from the ribosome is partly governed by its GTPase activity.” GTPase refers to the factor that pays the energy currency for the operation. The entire binding, cleavage and release sequence normally takes about 3 seconds. Here’s a summary of the process:
First, a prebound ternary complex of eRF1, eRF3, and GTP rapidly binds to a ribosome halted at a stop codon. eRF3 appears to unlock eRF1 conformation to facilitate fast ribosomal binding, because the association of eRF1 alone is slow and governed by an eRF1 concentration–independent event…. Next, eRF3 hydrolyzes GTP to promote its own release, which permits the rearrangement of eRF1 to an active conformation. Accommodated eRF1 then rapidly cleaves the peptidyl-tRNA bond, triggering ribosomal intersubunit rotation, movement of the deacylated P-site tRNA to a P/E hybrid state, and ejection of both eRF1 and the liberated peptide.
The termination process resembles translation elongation (the insertion of a cognate tRNA in the A site during translation), except that the GTP-bound eRF3, bound to eRF1, acts like a switch. The complex binds to the stop codon in the A site like a cognate tRNA would, but then eRF3 hydrolyzes its GTP which makes it self-eject. This, in turn, unlocks a conformational change in eRF1 that cleaves the polypeptide. The conformational change also rotates the ribosome, leading to ejection of eRF1 and the liberated polypeptide, soon to become a protein. That’s the short story. There may be more going on:
With the critical caveat that termination may also be influenced by unidentified nascent chain dynamics and other trans-acting factors, we propose that the termination mechanisms described here are fundamental to eukaryotic translation, because the release factors are widely conserved from yeast to humans.
Choreography Essential in All Life
This “essential process,” they note, requires the functioning of interdependent events. The “e” in eRF1 and eRF3 means “eukaryotic” because bacteria, too, have analogous factors (RF1/2, RF3) that perform corresponding functions. Translation termination is therefore essential for all living things.
Additionally, safety at these cellular stop signs is partly ensured by the “choreography” of ribosome activity, the length of transcripts entering the ribosome, and controls over the availability of assisting molecular machines. They only experimented with short sequences which may not adequately mirror what happens in vivo.
The finding that termination (~4 s) is fast relative to initiation [~20 to 60 s] but somewhat slower than elongation [0.05 to 1.4 s per codon] suggests the existence of an intricate choreography that prevents the accumulation of ribosomes at stop codons. Consistent with this, ribosomal profiling in eRF1-depleted cells revealed a marked increase in queueing of ribosomes at stop codons.
Mistakes, in other words, may be rarer than what they encountered in vitro, because long transcripts “substantially outnumber” short transcripts and may act to prevent delays of the termination factors. Returning to our traffic analogy, a lawbreaker may have less opportunity to run a red light if the lanes are all full and moving smoothly.
The scientists call this another aspect of what is known as “kinetic proofreading” — the prevention of mistakes by motion.
The fidelity of translation elongation is driven in part by kinetic proofreading, in which EF-Tu/eEF1Apreferentially rejects noncognate tRNAs in two sequential steps to boost overall accuracy. Although the basis of termination fidelity is unknown, we consider kinetic proofreading a plausible model. eRF3 is essential for termination fidelity, because its inclusion boosts specificity by 2600-fold. Here, we show that eRF3 conformationally unlocks and delivers eRF1 to ribosomes (Figs. 2 and 4) and facilitates eRF1 accommodationin an eRF3 GTPase–dependent manner (Fig. 4), thus providing eRF3 with multiple opportunities to favor genuine stop codons.
It’s no wonder that they call this a “tightly regulated, multistep process.”
What Causes Stop Codon Readthrough?
The team found that certain cis-acting mRNAs of untranslated regions promote harmful stop codon readthrough. They do it by hindering the cleavage activity of eRF1, lengthening the time of binding from 3s to 7s. The delay allows other substances to stabilize inappropriate tRNAs at the A site in the ribosome, preventing entry of the eRF1-eRF3-GTP complex. “Together,” they conclude, “these studies demonstrate that stop codon readthrough effectors hinder numerous facets of termination, thus uncovering additional nodes to target with potential therapeutics.”
In some cases of inherited diseases, doctors would like to promote readthrough. For instance, mutations that introduce premature stop codons are hard to treat. They result in unfinished polypeptides ejecting from the ribosome, unable to form essential proteins. They are intercepted as trash by other molecular machines that perform nonsense-mediated decay (NMD).
To achieve effective therapeutic readthrough of premature stop codons, elongation, termination, and NMD must all be carefully tuned to avoid widespread misregulation of gene expression while still eliciting enough readthrough to alleviate disease. Thus, termination and NMD inhibitors may prove most useful as adjuvants, lengthening the kinetic window for drug-mediated readthrough of premature stop codons.
Another Irreducibly Complex System
This short look at stop codons and how they trigger a coordinated series of actions in ribosomes reveals another irreducibly complex (IC) system at work. Stop codons keep us alive and healthy unless mutations, like lawbreakers, “run the red light” and violate the rules. What molecular biologists have uncovered since Darwin’s Black Box introduced the concept of irreducible complexity is not just an IC system here or there, but an IC set of IC systems at work. If one IC system defeats evolution, how much more a superset of IC systems? It’s important that people learn about the details of some of these systems to avoid being fooled by simplistic visions of cells emerging from primordial goo. Confidence in intelligent design comes when they say, “I see IC in the details.”