When you hear talk about the newest movie to hit theaters, your mind might go straight to an Oscar-worthy drama, or perhaps the rumored family-friendly feature starring a charismatic blue heeler. But today’s headline film is running on a different scale—one you won’t see reviewed on Rotten Tomatoes, yet it’s playing out inside every cell and helping scientists answer pivotal questions about how biology organizes protein production.
For decades, researchers have known that making the “right” protein starts with picking the correct start codon—typically AUG, the molecular equivalent of a starting line on the mRNA racetrack. Ribosomes usually pick this line with astonishing, single-nucleotide precision. Yet, for all this fidelity, biology sometimes bends the rules: ribosomes can launch from alternate, non-standard start lines, creating unexpected protein variants.
While AUG start sites are the most utilized, we’ve known since the 1980s that translation can start at non-AUG start sites. These alternate start sites most often differ by a single base such as CUG or GUG—and this doesn’t necessarily cause mistakes; rather it makes regulatory diversity. “When it’s off by a base, you don’t get a protein—or you might get the wrong protein, or even a toxic one,” explains translation enthusiast Dr. Chris Lapointe. Sometimes, he notes, “those have regulatory roles, and very often, they’re less efficient. You make less protein from those start sites.” But, in the context of protein synthesis, that’s sometimes the point.
To understand how the cell manages both single-nucleotide accuracy and adaptable regulation, Lapointe’s team in the Basic Sciences Division is using complex microscopy to image this process at the single-molecule level. “What we’re interested in is: how does the machinery have single-nucleotide precision but also use start sites that can differ by a single nucleotide? That’s the major question.” Using fluorescent labeling and purified human translation machinery, the team moved beyond static photos in a study recently published in Nature Structural and Molecular Biology. “In our field…it’s becoming pretty standard to have really nice photos and snapshots of all the initiation complexes. But…you want to put them in order and really understand the timing.”
The analogy that’s stuck with Lapointe: “It’s like a dinner party—someone’s snapping photos all night, but you just get a mixed-up deck of the snapshots. If you knew what the dinner was, you could probably order them. But if you had no context, it gets a lot harder. Our goal is to make a 3D animation, like a movie, rather than just a bunch of snapshots.”
The molecular movie revealed something startling. “The model going in…said both eIF1 and eIF5 are bound up to the start site, and then at the start site, eIF1 releases and eIF5 does a conformational rearrangement to take its spot. What we see, in humans, is that eIF5 is not bound at first. eIF1 stays bound, and when it hits the start site, it’s ejected. But then…eIF1 starts resampling. During that resampling period is when eIF5 tries to bind.” The result is a double-check: “If eIF5 successfully binds, you make a protein. If eIF1 successfully rebinds, the complex will start scanning again. So it’s like a double-check step.”
This nuanced choreography isn’t just academic. Differences in start site choice, and thus in which and how many proteins made, are magnified under cell stress, in cancers, and in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). “That ratio is really important,” Lapointe says, “and you can get disruptive ratios…which is why we care not just about efficiency but how frequency of AUG vs. non-AUG usage can change, depending on the cell’s context.”
Looking ahead, Lapointe and colleagues are pushing beyond the foundational “double-check” mechanism, focusing on new questions at the interface of molecular choreography and translational control. One immediate direction is to decipher whether eIF5, long viewed as a commitment factor, can also participate in downstream quality control—potentially proofreading ribosomal decisions for start-site selection.
But what about how this might play a role in human disease? The lab is also interested in investigating how molecular mimics of eIF5, including oncogene-derived analogs, may modulate translation in cancer cells, shifting the balance between canonical and alternative start codons and reshaping the proteome under pathological conditions. Another branch of research tackles repeat-associated non-AUG translation events in neurodegenerative disease, where aberrant initiation dynamics fuel toxic protein generation.
Finally, Lapointe’s team aims to map the feedback and cross-regulation between initiation factors themselves—a network that, when disrupted, could drive cellular adaptation or contribute to disease. By integrating single-molecule biophysics with genetic and cellular models, the group hopes to uncover new therapeutic entry points and reveal how the translation machinery rewrites its own rules in response to cellular challenges. Who’s ready for the newest feature film?
Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium Member Dr. Christopher Lapointe contributed to this research.
The spotlighted research was funded by the Howard Hughes Medical Institute Gilliam Fellows Program, the Stanford Bio-X fellowship, the Chan Zuckerberg Biohub Investigator Award, the National Institutes of Health, and the Damon Runyon Cancer Research Foundation.
Grosely R, Carlos Alvarado C, Ivanov IP, Nicholson OB, Puglisi JD, Dever TE, Lapointe CP. 2025. eIF1 and eIF5 dynamically control translation start site fidelity. Nature Structural and Molecular Biology. DOI: 10.1038/s41594-025-01629-y.
Science Spotlight writer Jenny Waters is a postdoctoral research fellow in the Hsieh lab at Fred Hutch. She studies how mRNA translation coordinates bladder cancer transformation and metastasis by post-transcriptionally regulating expression of oncogenic proteins. Outside of the lab, Jenny enjoys spending time with her dogs, convincing her husband to join her on trail runs, and pretending every steep hill is just a "gentle incline."
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