Live Blogger: Jennifer Baker 

Editor: Eilidh McClain

This piece was written live during the 7th annual RNA Symposium, “From Molecules to Medicines,” hosted by the University of Michigan’s Center for RNA Biomedicine. Follow MiSciWriters’ coverage of this event on Twitter with the hashtag #umichrna.

The “central dogma” of biology – that DNA is transcribed into RNA is translated into proteins – is a scientific tenet that haunts many American 10th graders during high school biology class. You might recall seeing diagrams like this one of an mRNA molecule sandwiched between the two halves of a ribosome as a new strand of amino acids unfurls from the exit site. 

However, it’s likely that your teacher didn’t spend much time on the how and why of this process – why does the ribosome bind to the mRNA? How does it find the start codon, the location on the mRNA that marks the spot where the ribosome starts translating? 

Don’t blame your high school biology teacher – until now, scientists didn’t even know! 

Answering this fundamental question has been a recent goal of Jody Puglisi’s lab. During today’s second keynote lecture, Jody is here in Ann Arbor discussing the result of this recent work, published in Cell in November 2022. 

“What animates me is animating biology” 

L’Étoile (The Star)
Edgar Degas, 1878

Jody begins with an analogy that he uses throughout his talk: the initiation of translation is like a ballet. Like Degas’ famous paintings of ballerinas, structural methods like cryoEM are useful for providing information about what each of the proteins involved – the “dancers” – look like at a particular point in time. What these structural images lack is temporal information – when each of the protein “dancers” enter and exit the stage, and the timing of the moves they perform relative to each other during the dance of translation initiation. 

To collect more information that a single frame can provide, Jody’s lab uses a technique called single molecular fluorescence resonance energy transfer (smFRET), which allows the “audience” of scientists to watch this molecular dance in real time. Like a film of Swan Lake compared to a Degas painting, smFRET brings the translation process to life, allowing Jody’s lab to track the speed, direction, and energy cost of the movements of each protein “dancer”.

A colorful cast 

To watch your own private showing of translation initiation with smFRET, you first need to cast the right dancers. While the pas de deux of translation is performed by the mRNA and the ribosome, there is a whole supporting cast of initiation factors like EIF4A that choreograph the precise way in which the ribosomal subunits find and “dance” with the mRNA. Because so many supporting “dancers” are required, this veritable ballet of proteins is quite slow compared to other molecular timescales, taking about 20 seconds for initiation to be completed. Also like the traditional pas de deux, elongation cannot begin until all other factors besides the mRNA and the ribosome leave, since the precise fit of the mRNA within the ribosomal subunits is hindered by the presence of any remaining initiation factors. 

Once the perfect ensemble of dancers has been cast, it’s time for colorful costumes that will stand out on the big stage. For real-time visuals of initiation, Jody’s lab borrows an approach from cellular biology: label each molecular dancer with fluorescent dye and watch their glow to see how and where they move. The fluorescent dyes used for smFRET transfer energy between each other when they are close in proximity, allowing Jody’s lab to track the location of molecules in space relative to each other. In addition, the duration of each burst of color gives clues to how long each of these interactions are. Together, these two features of smFRET allow Jody’s lab to determine the composition of the initiation complex and the conformation of individual factors at specific points in time. 

“Students, remember: a lot of science is entertaining yourself!”

After the dress rehearsal of benchmarking the smFRET technique, it’s finally time for opening night. Jody is certainly entertained by the performance, reminding the audience that “a lot of science is entertaining yourself”! 

The show opens ​​with a fast-paced number – loading the mRNA onto the small ribosomal subunit. This is a quick process and is highly energy-dependent – at least 10 ATPs are consumed! After this, a process called scanning occurs, where the initiation complex zips along the mRNA strand like a rollercoaster car on a track, looking for the AUG start codon sequence. Unencumbered, the initiation complex races along the RNA strand unidirectionally, starting at the 5’ end and proceeding towards the 3’ end, at a rate of 100 bases per second. 

hairpin structure in RNA
image source

However, there are a couple of features of RNA that can slow the scanning process down: hairpins and near-cognate codons. Hairpins are three-dimensional structures in RNA that form due to RNA base pairing with itself. Like actual hair pins, one strand turns back in a loop to base pair with itself in a parallel fashion, forming a section of the RNA that is double-stranded. To scan through a hairpin loop, the initiation complex must break the molecular interactions involved in base pairing and unwind the RNA so that it can fit through the ribosomal cleft, which is only a single strand wide. 

Near-cognate codons also slow the scanning process down, but for a different reason than hairpins. Near-cognate codons are sequences that are similar to the AUG start sequence but with a substitution at the first base position – for example, CUG, where cytosine is substituted for adenine. Because these sequences are so similar to the actual AUG start codon, the initiation complex often pauses on these codons to determine whether or not this is where elongation should begin. If the initiation complex encounters a CUG sequence before the correct AUG sequence, this decreases the likelihood of translation being initiated at the correct site by about 10%. 

Despite these hairpin and near-cognate codon obstacles for the initiation complex, it will ultimately settle on the correct AUG start codon in about 1 second! But before the large ribosomal subunit joins and the pas de deux begins, there is a long, awkward pause – 19 seconds long, to be exact! This is a long time on the stage and even longer on a molecular timescale, but this intermission is imperative for elongation. It takes every bit of these 19 seconds for all of the supporting initiation factors to leave the complex and allow the large ribosomal subunit to bind to the small subunit. Sometimes this process takes so long that if hairpins are present, they have time to reform and scanning must take place all over again! Even so, in a burst of fluorescence, the mRNA is eventually entwined within a catalytically active ribosome during the pas de deux – ready for elongation, the second act. 

Encore coming soon: from molecules to medicines

As Jody wraps up his talk, he ends on a slide with advice for the students in the audience. He emphasizes the importance of not losing the wonder of studying the inner workings of nature, since it is such a privilege to do so. He also urges students to learn the basics of biology early in their careers, which won’t go out of style and which will serve them well. 

After a flurry of questions from the audience, a coffee break, and another keynote, I caught up with Jody during the poster session. When asked about the translational potential of his research, Jody reiterated his interest in foundational biology. Though he likely won’t be working on clinical translation directly, Jody hopes that his lab’s work will catch the eye of people working in RNA vaccine development. Since this research focuses on what molecular factors affect efficient translation, he believes it has the potential to inform companies how to design an mRNA that can be efficiently translated by the host (e.g., considering hairpins and near-cognate codons during primary sequence design). He is currently working on the loading step of initiation, where the small ribosomal subunit is loaded onto the mRNA, a complicated and highly-regulated step in the initiation process. 

Like many in the scientific community, I look forward to an encore performance with new molecular dancers – or maybe even an adaptation for a clinical audience – soon!

About the speaker

Dr. Joseph “Jody” Puglisi is a professor of structural biology and director of the Magnetic Resonance Laboratory at Stanford University. Jody completed his bachelor’s degree in chemistry at Johns Hopkins and his PhD in biophysical chemistry at Berkeley before working as a postdoctoral researcher at both IMBC du CNRS (Strasbourg, France) and MIT. Jody started his professorship at University of California Santa Cruz before landing at Stanford, his current academic home, in 2004. He is a member of the National Academy of Sciences and is a leader in the field of RNA structure and function. His current research focuses on combining structural and real-time, single-molecule techniques to gain foundational insights into the mechanisms underlying RNA’s role in health and disease.

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