Fans of reality television shows about real estate such as House Hunters will recognize a common debate as a couple evaluates a property that seems to check all their boxes: One person is ready to close the deal, but the other wants to keep looking.
In human cells, factories called ribosomes must make similar decisions about where to set up shop on a strand of messenger RNA and begin constructing a protein, the molecules that do all the cell’s work.
They say real estate is all about location, location, location and that’s especially true in cellular biology. If the ribosome picks an even slightly incorrect location to start translating the mRNA strand, the result could be junk or even toxic. Faulty start locations also play a role in cancer and other diseases.
Ribosomes typically begin at the first start site that checks all the boxes. However, sometimes they skip that one and choose an alternative that is less efficient for making proteins but may serve a different purpose, such as regulating how the protein is made.
A pair of helper proteins influences how strictly ribosomes adhere to the standard checklist. But until now their interactions happened behind the scenes, like a couple’s off-camera argument on House Hunters before the big reveal of which property they finally chose.
A recent study published in the journal Nature Structural & Molecular Biology uses some innovative lab techniques to essentially eavesdrop on the behind-the-scenes argument between helper proteins in real time as ribosomes select their start sites.
Fred Hutch biochemist Christopher Lapointe, PhD, and his colleagues discovered that the debate between “Let’s commit!” and “Keep looking!” is much more spirited than previously understood.
The study advances our understanding of how breakdowns in that dialogue can lead to something far worse than picking the wrong house.
Messenger RNA is a go-between molecule that copies sequences of DNA code within the cell’s nucleus and then transports them to ribosomes outside the nucleus, where they serve as the instructions for building proteins.
When the process begins, the ribosomes are separated into a small piece and a big piece.
The small piece first attaches to one end of an mRNA and then a molecular initiation complex races along the strand, searching for the right place to set up shop. The initiation phase ends when the complex lands on a start site, signaling to the bigger piece of the ribosome to join. The united pieces of the ribosome lock onto the mRNA strand like pursed lips slurping a noodle.
Ribosomes translate mRNA code, which is composed of molecules called nucleotides that come in four flavors: Adenine, Guanine, Cytosine and Uracil. Those nucleotides are strung together on the mRNA strand to form groups of three, which are called codons that are identified by the first letter of each nucleotide in the group.
Each three-letter codon corresponds with a specific amino acid, the building blocks of proteins.
The ribosome slurps the mRNA strand one codon at a time, matching each three-letter codon with its corresponding amino acid, which is delivered “just in time” to the ribosome.
As the ribosome moves down the strand translating the codons, the resulting amino acids link together in a chain. When the final codon is translated and the last link is added, the ribosome has completed translation. The newly formed amino acid chain floats away, undergoing some additional changes and often combining with other chains before finally folding into a functional protein.
But first the ribosome must decide exactly where to begin translation.
The initiation complex moves along the strand and usually selects the first AUG (Adenine, Uracil and Guanine) codon it finds to begin building the protein.
The ribosome also must set the register for the entire translation by choosing which nucleotide within the start codon will be the first letter of the first codon translated into the first amino acid in the chain.
“If the register is off by a nucleotide in either direction, you don't get the right protein anymore,” said Lapointe, who works in Fred Hutch’s Basic Sciences Division. “You probably don't even get a protein. It's probably complete junk.”
Even worse, it might make something deadly for the cell.
Yet sometimes the ribosome loosens the rules about where to start, and Lapointe wanted to figure out the mechanism that enables that tradeoff between precision and flexibility.
The usual start site is AUG, but sometimes the ribosome chooses a different codon to begin translation, such as CUG, UUG or GUG . This flexibility helps control several variables, including how much protein gets made and where the protein goes in the cell.
Lapointe focused on two helper proteins called eukaryote initiation factors — eIF1 and eIF5 — which affect how strictly the ribosome will stick to its AUG checklist.
The simple story is that eIF1 binds the initiation complex first while it is scanning and drops off when the complex finds AUG. Then eIF5 takes its place and essentially closes the deal to start translation.
Lapointe wanted to see exactly how that handoff between the helper proteins occurs, which is difficult to observe in real time within a live cell.
“In one cell, there are about 10 million ribosomes and there are maybe 300,000 different messenger RNA molecules,” Lapointe said. “So, you can imagine you have many translation events happening on many different mRNAs. They have many different features that might all be changing.”
To observe how ribosomes decide where to start, Lapointe and his colleagues used several techniques to make a synthetic model of the key molecular components involved, isolated from the typical cellular activity.
The helper proteins and other components were labeled with special light-sensitive dyes that caused them to blink on in different colors when they get close enough to each other to bind. That blinking signal enabled researchers to track the activity of each individual helper protein during the initiation process, which could be recorded under a microscope as a video in real time.
“That allows us to use fluorescence microscopy to then watch initiation as it occurs on individual mRNA strands,” Lapointe said. “You have complete control, and you literally watch the factors as they bind and release and assemble the ribosome at the right spot.”
When they tracked the fluorescent-tagged helper proteins, they confirmed some expected things that proved their out-of-the cell model worked, but they also discovered some surprises.
They expected eIF1 to bind only once as the scanning complex moved along the mRNA strand and then release when it found AUG. They also expected eIF5 to take over, sealing the deal that allows the bigger piece of ribosome to lock on and begin translation.
But they were surprised to see something else happening.
Instead of dropping off when AUG was located, eIF1 came back and tried to re-bind several times, muscling eIF5 out of the way. Like the couple in House Hunters, eIF1 was arguing with eIF5 about whether to keep looking even though they had found a site that checked all the boxes.
Lapointe and his colleagues knew that under different stress or disease states, different start sites can modify the protein, slightly shortening or lengthening it to tweak its function.
By tracking the fluorescent-tagged molecules in real time, they could watch the behind-the-scenes debate, which acted like a dial modifying how strictly the ribosome would follow the checklist.
If the AUG start site is the one that’s needed, eIF5 displaces eIF1 and kicks it out for good so the rest of the ribosome can lock on and make a protein.
But if eIF1 overwhelms eIF5, the scanning complex starts moving again along the mRNA strand, resuming the search.
“This competition at that start site explains why the changes in the levels of either factor can change whether it's AUG versus non-AUG because these proteins are binding and releasing and those types of reactions are dependent on concentration,” Lapointe said. “Think of it like a dial. If you change the levels, you can tune whether you're going to be more AUG versus non-AUG.”
A well-functioning strictness dial is critical to the cell’s integrated stress response. A better understanding of how that dial works could shed light on what happens when it doesn’t work well.
“There's lots of hints that dysregulated AUG versus non-AUG initiations also play a role in human diseases as well, for example like in cancers,” Lapointe said.
This work is supported by the Howard Hughes Medical Institute Gilliam Fellows Program, a Stanford Bio-X fellowship, a Chan Zuckerberg Biohub Investigator Award, the National Institutes of Health, the Damon Runyon Cancer Research Foundation and the Proteomics and Metabolomics Shared Resource of the Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium. Christopher Lapointe, PhD, was a Dale F. Frey Scientist of the Damon Runyon Cancer Research Foundation.
John Higgins, a staff writer at Fred Hutch Cancer Center, was an education reporter at The Seattle Times and the Akron Beacon Journal. He was a Knight Science Journalism Fellow at MIT, where he studied the emerging science of teaching. Reach him at jhiggin2@fredhutch.org or @jhigginswriter.bsky.social.
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