Rachel Green greets the day before the sun creeps over the horizon. Even as her grad students and postdocs swill coffee to rouse themselves to semi-alertness, Green has been up for hours. She starts with a 5:45 spin class at the same gym where Olympic phenom Michael Phelps used to train, and then races to her lab on the seventh floor of the aging high-rise on the Johns Hopkins Medical Campus in Baltimore.
Every morning, she strides past the long, black lab benches cluttered with test tubes, pipettes, and smartphones as she moves from bench spot to bench spot, student to postdoc to student. She asks about the day’s plans, examines data to weigh in on findings, asks questions, laughs, and strategizes the next steps. Ideas and advice flow rapid-fire. “Try talking to my colleague,” she advises one grad student. “Maybe run another trial,” she says to another.
“She’s a whirlwind,” says PhD student Anthony Schuller, as he frantically finishes, beneath an inflatable T. rex dangling from the ceiling, one last set of experiments to include with his thesis.
With morning rounds complete, Green retreats to her sparse but neatly organized office. It is the nucleus of the lab, and at the heart of the nucleus is Green herself. As always, the door is propped ajar. Clues to her lifelong obsessions – the molecule RNA, the synthesis of proteins, and the origin of life – adorn the walls: a framed page of an aged study describing the protein-making ribosome, and several posters detailing the familiar cloverleaf shapes of tRNA, the molecule that brings amino acids to the ribosome.
Understand the ribosome, she says, and you can understand the beginning of modern-day biology.
The ribosome, to Green, sits at the very center of life as it exists, and how it evolved in the first place. Understand the ribosome, she says, and you can understand the beginning of modern-day biology. Recent studies have pushed back the origins of life as we know it to 3.8 billion years ago, only 800 million years after the chunk of rock we call home congealed and cooled into a solid mass. But long before DNA’s double helix encoded information that other molecules would then use to make proteins, the related molecule RNA did both. Today’s ribosome is a complex cluster of molecules that work side by side to stitch proteins together, amino acid by amino acid. Its outsides are coated with proteins, added during billions of years of evolution, but RNA sits at its center. And that RNA is key to the ribosome’s function.
To crack the workings of the all-important ribosome, Green, an HHMI investigator, has had to become a catalyst herself, corraling techniques and scientists from disparate fields to get at the answers. This, as much as anything else, is Green’s specialty. Bring together the right elements under the right conditions, and “chemistry happens,” she says.
Green originally aspired to pursue a career in engineering as an undergraduate at the University of Michigan. That ambition did not last long. In her first engineering class, “they told me what pencil to use and where to write my name, and I got a claustrophobic feeling,” she says. “I went to the chemistry department and changed my major that day.”
She was accepted into a PhD program at Harvard University in 1986 and wound up in the lab of Jack Szostak (now a Nobel laureate and HHMI investigator), where she spent the next five years studying the evolution of a type of RNA called a ribozyme. Unlike its double-stranded cousin DNA, RNA is single stranded and also a wildly versatile molecule. Like DNA, it carries genetic information, but some kinds – ribozymes – can also catalyze chemical reactions, as proteins do. Because of this dual ability, they were thought to play an important role in the evolution of early life. Green’s RNA/ribozyme interest continued throughout her postdoc, when she moved across the U.S. to Harry Noller’s lab at the University of California, Santa Cruz, to investigate the function of the ribosome (the ultimate ribozyme) in the bacterium Escherichia coli for the next five years.
“The ribosome is the beginning of modern-day life. There were no proteins, as we know them, before the ribosome. It’s the bridge. It’s what took us from the RNA world to the protein world. And it’s unambiguously made of RNA,” Green says today, leaning back in her desk chair and lacing her fingers behind her head. Even after 30 years, her enthusiasm for the topic hasn’t dimmed.
Advisors, students, and colleagues alike note the passion and curiosity trapped in Green’s petite frame that continue to drive her work. “She’s bubbling with intellectual energy, and she is a very rigorous thinker. When she is satisfied with a study, you can be sure it’s done,” says Noller.
By the time Green began work on the ribosome in earnest, scientists had figured out the major details of how it turns instructions carried in messenger RNA (mRNA) into proteins. The ribosome has two subunits made of protein and ribosomal RNA that come together when the ribosome is active, like two buns of a burger. This ribosomal burger slides along a piece of mRNA, and the structure of the protein-to-be is specified by the order of nucleotides of the mRNA. Each grouping of three nucleotides – called a codon – dictates what amino acids, in what order, should be added to a growing protein. The amino acids are brought in and matched to the right spot by transfer RNAs, those cloverleaf-shaped structures gracing the posters in Green’s office. Ribosomal RNA then catalytically links the amino acids together.
In the mid-1990s, when Green joined Noller’s lab, the ribosome and protein synthesis – a process known as translation – appeared to have been mostly solved. Excitement for the Human Genome Project was growing, and many scientists had turned their attention away from the problem of protein formation toward the nuts and bolts of gene regulation and synthesis of mRNA. But Green sensed there were ribosome details still to be filled in, proteins and factors yet to be identified that could fine-tune the rate at which a gene’s encoded protein was made and influence the ultimate expression of a gene. She hammered out more details of just how E. coli ribosomal RNA oriented the tRNAs to catalyze the reactions that brought amino acids together. Then she carried the ribosome work with her when, in 1998, she moved to the Molecular Biology and Genetics Department at Johns Hopkins as an assistant professor.
At Johns Hopkins, Green took a new tack: purifying defective “mutant” ribosomes gleaned from genetically deficient strains of E. coli to define their precise inner workings. She looked at the roles of various accessory proteins, for example, identifying two small protein subunits that are important for controlling the processive movements of the massive machine along the mRNA.
She also showed that one layer of the ribosomal RNA is critical to form the peptide bond and another layer to release the completed protein from the last tRNA molecule. The longer she worked, the more detailed her work became, until she found herself studying the catalytic site of the ribosome at the level of individual protons. “I hit the limit of what I could do and wanted to do,” she says.
She also wanted to broaden her network of colleagues beyond those working on catalysis in bacterial ribosomes to include those studying how ribosomes helped control the rate of protein synthesis and mRNA degradation. Jeff Coller, a biochemist at Case Western Reserve University in Cleveland, was one of those people. He and Green had been friends for a decade when they realized they had both zeroed in on the same protein that appeared to interact with the ribosome. They decided to collaborate. “She was very generous. This wasn’t someone who wanted to keep all the cookies to herself,” Coller says. “She’s not afraid to tap into others’ expertise.”
So after nearly a decade, Green moved from E. coli to yeast, a more complex organism that has evolved a more fine-tuned control of ribosome activity. What excited her the most were the proteins assisting the ribosome whose functions were wholly unknown, and the development of new methods to tackle that problem. Two other researchers, HHMI Investigator Jonathan Weissman and his then-postdoc Nick Ingolia, had recently developed ribosome profiling – a technique in which you could freeze translation mid-stream and then remove all of the messenger RNA that wasn’t currently tied up in a ribosome, leaving only those mRNA segments still nestled within it, which the researchers then sequenced. Green saw the power in this genomewide in vivo approach to help understand how a cell responds when translation goes awry.
This wasn’t someone who wanted to keep all the cookies to herself,” Coller says. “She’s not afraid to tap into others’ expertise.”
In an ideal world, the lopsided sandwich of two ribosome halves would slide smoothly along the mRNA, codon by codon, straight through from start to finish. But the process rarely goes perfectly. Caches of tRNAs run low. Bonds between amino acids struggle to form. Messenger RNAs have structural impediments. As a result, ribosomes often stall midway through translation. Through the use of ribosome profiling, Green’s lab identified problematic RNA sequences that are especially prone to stalls; these were overrepresented in the profiling results. Then they looked for proteins that rescued a stalled ribosome and helped resume translation.
Dom34, a protein the lab had studied biochemically for years, was critical in enabling stalled ribosomes to abandon ship and break apart into their two halves, allowing the cell to reuse the subunits and thus unclog the system. “Whenever the ribosome does something wrong, Dom34 cleans it up,” Green says.
She and Coller together studied a protein called Dhh1p. They showed that Dhh1p tells ribosomes when translation is moving too slowly, signaling the cell’s decay machinery to degrade the mRNA. Coller’s earlier work showed that mRNA codons not only tell the ribosome which amino acids to add but also how quickly to add them. Even codons that direct the addition of the same amino acid can have different speeds – and, potentially, profound effects on how easily a protein is made, and thus how abundant it is in the cell.
The rich insights gleaned from all this work owe a lot to Green’s personality and scientific style, colleagues say. “She loves people and she’s plugged into what’s going on now. Rachel can put her finger right on the problem and figure out what will have the most impact,” says Allen Buskirk, who shares Green’s lab space at Johns Hopkins.
In the last few years, Green has been studying ribosome function in human cells, including immature red blood cells called reticulocytes. Eric Mills, an MD-PhD student who split his time between the Green and Ingolia labs, performed ribosome profiling on reticulocytes and found that the untranslated tail-ends of the mRNAs were filled with ribosomes that had done their protein-making job but weren’t then removed from the mRNA. “It’s unlike the pattern in any other cell type,” Green says. “We studied for years the proteins involved in getting ribosomes off mRNA. We know how they work. And this is a cell type where it’s not working.”
It turns out that some of the protein factors that assist with termination are made in mitochondria, the cell’s energy generators. But reticulocytes discard their mitochondria, which Green thinks may explain why the cells are deficient in these factors. To get around this, reticulocytes seem to have extra Dom34 on hand to pry off some of those stalled ribosomes so they can make the hemoglobin protein that’s critical for these red blood cells. This may explain why diseases that affect ribosome function, known as ribosomopathies, disproportionately affect reticulocytes, explains Green: with so many ribosomes stuck on mRNA, the cells can’t tolerate even the slightest error.
Enthusing over her reticulocytes, Green has been talking for more than an hour in her characteristically clipped, rapid-fire style. But now she takes a rare, brief pause and leans forward over a notepad covered with blobby sketches of ribosomes and ripples of mRNAs. “So,” she says, “that’s what I’m most excited about this week. It could be different next week.” Then she adds: “Part of what makes this project fun is that these students are just so good. When you have a great student or a great postdoc, it’s really fun.”
Green’s idea of fun doesn’t stop at the lab, says former PhD student Luisa Cochella, who now runs her own group at the Research Institute of Molecular Pathology in Vienna. “Rachel is always the first person out on the dance floor at any meeting. In 2002, at the Ribosome Meeting at Cold Spring Harbor Labs, we all ended up singing ‘We love the ribosome,’” Cochella says. Green sang the loudest of them all.
And it’s a love still pounding strong a decade and a half down the road. “People have asked why I care so much,” Green says. “The ribosome is this elaborate, finely tuned, efficient machine. In all parts of my career, I’ve tried to understand the order and speed of the chemical reactions that make biology work. For the ribosome, understanding these parameters can tell us how the ribosome does its magic – how it decides what to translate, and how it accurately synthesizes the tens of thousands of wildly different proteins in each of us. And in the protein-based world we now live in, this is at the core of everything.” ■
Story by Carrie Arnold
Photography by Eli Meir Kaplan