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DNA melding into phylogenetic tree
How did life begin?
Protocell researcher and RNA expert bump into each other and learn about early life on earth
27 July 2020

Some things are alive and some things are not. Seems like an easy and obvious concept, but drawing the line that separates “alive” from “not alive” is not quite as straightforward as you might think. What makes something alive? Well, if you Google “properties of life,” you will see that there are seven (or eight, or nine, or maybe even ten) things that everyone “agrees” are key—things like growth, the ability to reproduce, response to stimuli and the environment, maintaining homeostasis, and, often, cellular organization.

So, a quick quiz.  A rock? Not alive. An oak tree? Alive. Water? Not alive. Single-celled bacteria? Alive. An RNA virus? Well, now things get interesting.

Most viruses are simple. They are composed of an outer shell or membrane that surrounds some genetic material: sometimes DNA, sometimes RNA, sometimes both. They generally can’t, however, reproduce on their own—one of the essential properties of life. Instead, they co-opt the reproduction machinery of the cells they infect, making the cell make more viruses. So, there is some disagreement among scientists about whether viruses count as alive or not, but because they contain genetic material, they are, at least, likely to be related to other life on Earth. All life that we know of shares the use of DNA or RNA to carry genetic information, which suggests a shared origin; viruses are somehow connected to the evolutionary tree of life—which brings us to a second line between life and no life.

If we think about the history of the Earth, at some point in the past there was no life and now the place is practically bursting at the seams with it. How did that happen? That is a question that has perplexed humanity for millennia. Two Eberly College of Science researchers have brought the diverse expertise of their labs together to explore some possible answers.

Life in its simplest form could be thought of as a collection of molecules that perform chemical reactions and that are somehow compartmentalized, isolating them from their environment. Christine Keating, professor of chemistry, studies physical chemistry and the self-assembly of molecules. As an undergrad she studied chemistry and biology, so she gravitated toward studying the materials chemistry of life and how cells might have evolved. Phil Bevilacqua, head of the Department of Chemistry and Distinguished Professor of Chemistry and of Biochemistry and Molecular Biology, has been studying RNA for nearly 30 years. RNA can both carry genetic information and perform chemical reactions, making it an attractive candidate for playing a role in early life.

“There was this old Reese’s Peanut Butter Cup commercial where one person had chocolate and another had peanut butter and they bumped into each other,” said Keating. “The chocolate got into the peanut butter and they were like, ‘This is amazing.’ I think of our collaboration like one of us had the chocolate and one of us had the peanut butter. Our research really complements each other’s to be able to look at possible scenarios for the evolution of early life.”

It’s an RNA world

Or at least it was. Maybe.

Phil Bevilacqua
Phil Bevilacqua

“How life actually began is basically unknowable,” said Bevilacqua. “There is no fossil record of the early steps that led to life, and we can’t go back to observe what was going on. What we can do, though, is use what we know about the Earth three to four billion years ago and what we know about the simplest forms of life to build models and test hypotheses. We can begin to narrow down what might have been possible and eliminate ideas that probably aren’t.”

One of the leading hypotheses about the origin of life on Earth leans heavily on the role of RNA. RNA is DNA’s lesser known little brother; it stands for ribonucleic acid (DNA is deoxyribonucleic acid). Both molecules are long strings composed of four basic subunits, and it’s the sequence of these units that encodes genetic information.

The four subunits of DNA are adenine, thymine, cytosine, and guanine; they are called nucleotides or bases, but we often just refer to them as A, T, C, and G. The DNA molecule is double stranded. Think of a twisted ladder: The side rails of the ladder are the two strands, and the rungs represent the bonds that hold them together. The two strands are complementary to each other: If there is an A on one side of the rung, the other side will have a T; and if there is a C on one side, the other will have a G.

RNA shares three of the four subunits with DNA but has uracil (U) instead of thymine (T) and is single stranded. Because sections of the RNA molecule can be complementary to other sections, the molecule can fold up on itself, bonding in a way similar to double-stranded DNA, with U complementing A. The resulting three-dimensional structures of RNA molecules can allow it to do more than just encode information; it can carry out functions that are nowadays mostly associated with proteins.

“RNA became implicated in early life scenarios in the late 1960s,” said Bevilacqua. “It was recognized that the complex structures that RNA forms could potentially perform catalysis—it could drive chemical reactions. So, you have a single molecule that sort of solved the ‘chicken and egg’ question of which came first in the origin of life: genetic information or catalysis? Work in the 1980s that led to a Nobel Prize demonstrated that RNA can in fact do both, so it became a prime candidate for how early life could have evolved in what is known as the RNA world hypothesis.”

The research in Bevilacqua’s lab focuses on understanding the functions of RNA at the molecular level. His group studies the mechanisms of RNA enzymes—RNA molecules that have functions similar to protein enzymes in chemical reactions—known as ribozymes. Their research helped to establish the role played by ribozymes in proton transfer and RNA cleavage, and they are currently studying the role of metal ions and other cofactors in these mechanisms. They also study the precise mechanisms of how RNA molecules fold in cells. Studying the role of RNA in early life came later.

“Because I work on RNA catalysis, I’ve always been interested in its potential role in early life, but I never took it too seriously,” said Bevilacqua. “It would come up as the last sentence of a discussion in a paper: ‘This versatility of RNA to perform reactions could relate to the RNA world hypothesis.’ But in thinking of what it would take to go from a bunch of molecules floating around with the ability to perform reactions to something that could be called early life, I started to work with Chris, putting together her expertise in protocells with my expertise in RNA.”

Compartmentalize it!

Chris Keating
Christine Keating

If you think about a modern cell with its hundreds of components—organelles, proteins, DNA, and RNA—working together seamlessly to perform its varied functions, it can be hard to imagine how it could have evolved. But we know that it must have. Before there were modern cells, there had to be earlier, simpler ones. And before that, there had to exist the components to form them.

We can imagine an early, prelife Earth in which chemical reactions are occurring that begin to produce molecules that resemble things like RNA. But even if these early molecules could potentially encode genetic information and perform catalysis, they would be floating around in the proverbial primordial soup, and the chance they would find each other and actually do these things seems incredibly slim. A necessary step, therefore, in the early evolution of what would become life was to increase the chances that these molecules would come together where they could begin to interact in more and more-complex ways. One way to do that was to compartmentalize them.

Coacervate droplets with multiple compartments
False color image showing how liquid-liquid phase
separation can form multiple compartments in coacervate
droplets. Credit: Keating Laboratory

“We were studying the self-assembly of molecules, what molecules can do on their own, and it’s kind of amazing what you get for free,” said Keating. “An example is lipid self-assembly. Lipids are fatty compounds that are insoluble in water. If you have some lipids and you add water—boom! Vesicles form. It’s amazing to watch.”

Among other things, Keating’s group studies artificial cells. Somewhat counterintuitively, she decided to start by recreating the cell’s cytoplasm—the goo on the inside that is often ignored—rather than its membrane. Even without membranes, you could get compartments through liquid-liquid phase separation.

“We always knew that if we were going to make artificial cells, it needed to be in a way that we could learn about real cells,” said Keating. “So, starting with the cytoplasm—where everything within the cell takes place—made sense, even though people may think it’s the boring part. We were kind of amazed at how quickly things got interesting.”

Macromolecules (big molecules—like RNA, for example) would concentrate into different liquid phases within the artificial cytoplasm.

“Without really trying, we found a solution to one of the big problems in studying the origin of life, which is, ‘How do you put stuff in your compartment?’” said Keating. “You can make a membrane really easily, like with lipids, but it’s incredibly difficult to get things inside of them. With our system, you get these phase-separated droplets—called coacervates—in which macromolecules collect, and you can concentrate them from a really dilute solution. What better to study in this system than RNA, which is where Phil comes in.”

Two great tastes that taste great together

Coacervate droplets, with inset showing RNA molecules concentrated inside the droplets
Microscope image of coacervate droplets. Inset shows
fluorescently labeled RNA concentrated in the droplets.
Credit: Bevilacqua Laboratory

“Working with Chris, we really started to think about how it all began, how did life start, how do you compartmentalize molecules to facilitate the types of reactions we were studying,” said Bevilacqua. “We began to look at what’s possible, and not only that but what would be feasible and probable. I was coming from one angle and she was coming from another, and there was a confluence of our two approaches that really made sense.”

The collaboration is already paying dividends. Recently, the two groups published a paper demonstrating that not only could RNA be assembled from its constituent parts inside of membraneless compartments but the concentration of the RNAs in the compartments also enhanced the enzymatic activity of ribozymes.

Whether or not these particular steps actually took place during life’s evolution on Earth, the collaboration of Keating’s and Bevilacqua’s research groups is advancing our understanding of what might have happened. They go together like chocolate and peanut butter.  

“I’d like to lay claim to being the chocolate in the Reese’s cup,” said Keating.