Christine Dunham is a leading expert on the ribosome — an elaborate macro-molecular machine that operates like a factory within a cell to manufacture proteins.
“The ribosome is really the most fascinating molecule in a cell,” says Dunham, Emory Samuel Candler Dobbs Professor of Chemistry. “It’s the hub of every activity. If you’re not making proteins, you’re not able to grow, function or communicate.”
Most of the time the ribosome protein factory runs smoothly and to plan. Sometimes, however, a process known as “frameshifting” can cause operations to go off script.
In her latest paper, published in Nature Communications, Dunham and colleagues showed through advanced imaging techniques how a small RNA chemical modification, known as a methyl group, influences gene expression. Specifically, they found that transfer RNAs, cause messenger RNA frameshifting on the ribosome.
Visualization of this process helps confirm a long-standing hypothesis for transfer RNA’s role in frameshifting, as well as providing detailed data on the exact molecular mechanisms involved.
“There is so much built into the ribosome to prevent it from making mistakes,” Dunham says. “It’s kind of shocking that the removal of a single, tiny methyl group leads to defects that cause this machinery to lose accuracy in protein synthesis.”
The findings help lay the groundwork for advances in synthetic biology to both treat diseases and to develop preventative therapies.
First author of the paper is Evelyn Kimbrough, who did the work as an Emory PhD in student in Laney Graduate School. She graduated in 2024 and is now an AAAS Science and Technology fellow at the National Center for Advancing Translational Sciences.
Co-authors include Ha An Nguyen, who graduated with a PhD in chemistry from Emory in 2021; Jacob Mattingly, an Emory postdoctoral researcher in the Dunham lab; and scientists from Columbia University, Thomas Jeffereson University and City University of Hong Kong.
Keeping cells humming
Proteins are the machines that make cells run. They perform diverse functions related to immune defense, nutrient storage, cellular communication, regulation of gene expression and growth and development. They work in concert with the nucleic acids DNA and RNA that store the blueprints for life.
In fact, the ribosome is made mostly of RNA, which does not just store information but can also act as an enzyme, catalyzing chemical reactions. The ribosomal RNA gets instructions for performing these reactions by “reading” what is known as messenger RNA (mRNA).
The ribosomes normally move along an mRNA strand and read specific sets of three nucleotides at a time, known as a “reading frame,” or “codon.” This mRNA codon is read by the corresponding three nucleotides of the what is known as the “anticodon,” located in transfer RNA (tRNA).
In some cases, however, the ribosome can stall, which may allow movement that deviates from the normal reading frame, a process known as “frameshifting.” Instead of reading three nucleotides at a time, the ribosome is reading two or four nucleotides, altering the message.
Sometimes frameshifting helps to control the levels of specific proteins to ensure proper function. More often, however, frameshifting is a negative effect that garbles the message and can lead to disease.
Setting the stage for new therapeutics
Dunham’s lab is working to visualize the mechanics and tease apart the effects of various RNA chemical modifications on the function of the ribosome.
For the current paper, the researchers used single-molecule fluorescence resonance energy transfer imaging, in collaboration with researchers at Columbia University, and cryogenic electron microscopy. This combined approach allowed them to better understand how a single methyl group appended to guanosine at a particular position in tRNA affects frameshifting.
“For the first time, we’ve shown at the molecular level how this particular chemical modification controls the ribosome,” Dunham says.
“If we can reduce the fidelity of the ribosome when we remove a single methyl group, that means we may also be able to manipulate and engineer the ribosome,” she adds. “We’re providing tools to expand the capabilities of doing synthetic biology for human health.”
For example, bioengineering may be able to reverse genetic mutations that cause frameshifting and lead to disease. Understanding how ribosomal frameshifting occurs may also help improve the development of antibiotics, vaccines and other therapeutics.
In 2018, Dunham and colleagues published a paper in the National Academy of Science’s journal PNAS, mapping how insertions to a tRNA also cause mRNA frameshifting. These frameshift suppressor tRNAs are now being used as therapeutics to prevent premature stop codons — mutations that cause a protein-encoding gene to stop producing protein earlier than normal, leading to human diseases.
That paper won the journal’s Cozzarelli Prize for that year, celebrating “scientific excellence and originality.” A commentary in PNAS called the work a “culmination of a half-century quest,” since the mutation in other tRNAs that suppress frameshifts were identified in the 1960s, yet the molecular mechanism was not substantiated until recent years.