Evolutionary Biology

Origins of Life II: Primeval environments and the origins of RNA

Did you know that scientists don’t need time travel to mimic conditions on Earth 4 billion years ago? Scientists who want to experiment in an environment like that of primordial Earth need only to visit volcanoes, which have chemical conditions similar to those on Earth long, long ago. That’s just what chemist David Deamer did in his research into the origins of life. Just as had happened in the lab, Deamer’s volcano experiments produced a necessary step toward the formation of living matter.

Beginning with Stanley Miller’s famous experiment in 1952, origins of life researchers have shown that all major classes of life’s chemical building blocks can form spontaneously under conditions found on the young Earth. While sugars, amino acids, and fatty acids are necessary for abiogenesis (the spontaneous emergence of life) to occur, they are not all that is needed for life to happen. Another big jump must occur: They must form long polymers and fold into complex shapes. These large molecules made of small building blocks are called macromolecules.

Some macromolecules are important for their catalytic abilities, which means they facilitate chemical reactions that cannot occur without help. These catalysts are called enzymes and are usually made of protein, although on early Earth the first catalysts may have been made of RNA. Other molecules are important because they store and process genetic information so that it can be retained and passed on for generations. Like letters of the alphabet linked into words, small building blocks such as amino acids and nitrogenous bases can hold information only when they are linked together to form large polymers.

While organic building blocks like sugars, amino acids, fatty acids, and nitrogen bases formed easily in multiple environments found on the primeval Earth, polymerization into functional macromolecules was a little trickier and certainly took more time. Experiments, like the one conducted by Tracey Lincoln and Gerald Joyce in 2009, tell us that groups of RNA molecules can grow, sustain one another, and even evolve, all in the complete absence of protein enzymes (see Origins of Life I: Early Ideas and Experiments for more details on this experiment). This is key to our understanding of how the chemistry of life may have started, and most scientists support the notion that RNA was the first large functional macromolecule to have appeared on early Earth.

There is just one problem, however. The experiments that show that catalytic RNA molecules can form, copy each other, and evolve all take place in test tubes. A confined space is crucial for these reactions to occur so that the molecules can accumulate to a high concentration, bump into each other frequently, and perform chemical reactions on each other. There were no test tubes on early Earth, so how could the RNA enzymes have ever formed in the first place? Once formed, the small building blocks would simply diffuse away from each other and be diluted into the vast depths of the ocean.

But nature can make its own version of a test tube: Our cells are tiny compartments, separated from the outside environment by membranes, which serve as barriers, like the glass around a tube. Membranes are made of lipids, which existed on the ancient Earth, alongside amino acids, sugars, and nitrogenous bases. For this reason, abiogenesis is linked to the question of whether membranes might have formed spontaneously from lipids in Earth’s primeval environments. If so, a drop of ancient water containing building blocks of proteins and RNA would also have contained trillions of tiny membrane-bound compartments. Holding the building blocks inside, those compartments would have acted as nature’s laboratories, and this scenario would have been vital to the origin of living cells.

Spontaneous formation of biological compartments

In the early days of abiogenesis research, the emergence of membranes sounded like a chicken and egg problem. Scientists understood that modern cells build and maintain their membranes using protein enzymes; however, without membranes, there is no compartment in which enzymes can be built. Without enzymes, there is no way to make membrane lipids. Also, modern membranes include various proteins interspersed with the lipid molecules. These membrane proteins perform a variety of specialized functions, such as catalyzing biochemical reactions and acting as “gates” to permit specific molecules and ions into and out of the cell (read our modules Membranes I: Introduction to Biological Membranes and Membranes II: Passive and Active Transporters to learn more).

Early membranes must have been very different from modern ones. Unlike the complex systems of lipids and proteins that comprise living membranes, simple kinds of membranes can actually form spontaneously and may have been forming all over the primeval Earth.

Anybody who makes bubbles with soap has seen the tendency of lipid molecules to form spherical shapes when they are in contact with water (see our Lipids: An introduction module). Soap molecules are amphipathic, meaning they possess both a water-loving and a water-hating portion. Each molecule has a “head” section that is hydrophilic (water-loving), because it contains polar covalent bonds using atoms such as oxygen, nitrogen, and phosphorus. Each molecule also has a “tail” that is hydrophobic (water-hating), because it consists of nonpolar bonds of only carbon and hydrogen atoms (Figure 1; see our Membranes I: Introduction to Biological Membranes module to learn more).

Figure 1: A phospholipid. Note the hydrophilic, or water loving, "head" section and the hydrophobic, or water hating, "tail" section.

image ©OpenStax College

The simplest amphipathic molecules are a type of lipid called a fatty acid. Each fatty acid molecule is a hydrocarbon chain with a carboxyl (COO-) group attached to one end. This charged group is very soluble in water. However, the hydrocarbon tail, with its many nonpolar C-H bonds, is very hydrophobic. This is why fatty acids are useful in making soap. They can dissolve oils using their hydrocarbon tails, but are still soluble in water because of their COO- head groups. These two abilities allow soap to break up oil deposits and stains and then to allow the oil to be washed away with water.

It has been known since the 1960s that fatty acids in a watery solution will be attracted to each other and form spherical structures called micelles. The shape of the micelle tucks the hydrophobic tails together, away from the water solvent (Figure 2).

The COO- group of fatty acids can also serve as an attachment point for connecting with other molecules, leading to much more complex amphipathic molecules with extremely hydrophilic heads. Phospholipids are the complex amphipathic molecules that make up cell membranes today. Living cells use enzymes to build these phospholipids out of fatty acids, glycerol, and phosphate as substrates. Although all of these building blocks existed on Earth prior to life, it was not known if they could spontaneously form into phospholipids without the help of an enzyme.

Comprehension Checkpoint
The earliest membranes on Earth were most likely just as complex as modern membranes.

Testing lipids from Earth and from space

In 1977, chemist David Deamer mixed glycerol, phosphate, and several different fatty acids in a test tube. When he put this mixture under primeval conditions (a mixture of the gases thought to exists in Earth’s primeval atmosphere), he found that phospholipids emerged spontaneously.

This was a key discovery because phospholipids will then spontaneously self-assemble into three-dimensional membranes whenever they are placed in water. Just as fatty acids are attracted to each other and form micelles under most conditions, phospholipids are attracted to each other and form membranes. The difference is that the membranes are called a “bilayer” because the phospholipids arrange themselves in two rows facing back-to-back. Like the micelles, this tucks the hydrophobic tails inward, away from the water (Figure 2).

Figure 2: Three of the different structures phospholipids can form in an aqueous solution: micelle, liposome, and bilayer sheet. In this depiction, the hydrophilic heads are round and white and the hydrophobic tails are yellow wavy lines.

In his experiment, Deamer also included amino acids in the mixture. The amino acids didn’t contribute to the formation of membranes, they were included to see if some of them would be “trapped” inside the membrane compartments when they spontaneously formed. They did. This was a crucial discovery because it showed that the early building blocks found on primordial Earth could spontaneously give rise to membrane spheres and that these spheres would randomly trap nearby molecules inside them. These structures are called liposomes and many believe that they were the ancient ancestors to living cells.

While this was a very encouraging finding, phospholipids are fairly complex molecules and Deamer needed to use the ingredients at very high concentrations to get them to form liposomes. Many have doubted that these molecules would have been found at such high concentrations on early Earth.

Hope of resolving this conundrum literally fell out of the sky in 1969 when a meteorite landed in the town of Murchison, Australia (Figure 3). A piece of this meteorite was delivered to NASA’s Ames Research Center in Mountain View, California, and scientists began to analyze its chemistry. One NASA scientist, George Cooper, discovered sugars in the meteorite, while others discovered amino acids, not just the 20 found in life on Earth, but 70 different kinds. Being a professor at nearby University of California at Davis, Deamer had colleagues at Ames and eventually he obtained his own sample of the meteorite on which to conduct his own tests.

Figure 3: The Murchison meteorite, which landed in Australia in 1969, has been shown to contain many types of chemicals required by life on Earth. On the right is a pebble-sized fragment of the meteorite; when magnified 10 times and placed in polarized light, a slice of the meteorite reveals various minerals in different colors.

image ©NASA

Deamer began by grinding up a small part of the meteorite and performing an organic extraction to pull any hydrophophic compounds out of the dust. Then he put those organic molecules into water, to see how the molecules would behave. This was a vital experiment, since the molecules extracted from the meteorite were as ancient as the meteorite itself. They were molecular remnants of the solar system when it was young, four billion years ago, when the same kind of molecules were being delivered to a primordial Earth that was also accumulating water from comets.

Immediately, the material coalesced to form micelles and membrane liposomes. Analysis of the membrane-forming compounds showed that they consisted of various fatty acids, with hydrocarbon tails of varying lengths. While fatty acids are not as efficient as phospholipids at forming membranes, their hydrophobic tails do gather together spontaneously and create tiny compartments, either micelles (hydrophobic tails fill inside of the sphere), or liposomes (spheres surrounded by a lipid bilayer like a cell).

Whether free fatty acids in water form micelles or liposomes depends on various chemical conditions in the aqueous solution where they are placed, most importantly, the pH. COO- groups in fatty acids are usually un-protonated (that is, they do not have a hydrogen (H) atom attached). But under acidic conditions, a proton will join to form COOH instead. For any mixture of free fatty acid molecules, like those extracted from the Murchison meteorite, as the pH of the environment drops, more and more of the fatty acid molecules pick up a proton so their carboxyl groups go from COO- to COOH. This makes the carboxyl less hydrophilic, making it harder to form a bilayer, so the fatty acids form micelles instead. On the other hand, free fatty acids form bilayers very easily to create liposomes at pH ranges between 7 and 9, depending on the exact type of fatty acid. By tweaking factors other than pH in his solutions, Deamer was able to get liposomes from the Murchison fatty acids, even at low pH.

Comprehension Checkpoint
When phospholipids are placed in water, they spontaneously form

Utilizing primeval environments on Earth

Given that organic solvents were abundant on early Earth, as was water, and that meteorites bombarded Earth much more often than they do now, it appears possible, likely even, that Earth received membrane-forming materials via meteorites. Whether the fatty acids formed naturally on Earth or came from a meteorite, the formation of membranes was a crucial step in the origin of living cells.

Without time-travel, we’ll probably never know the precise order of events and source of the biomolecules that gave rise to cells. However, Deamer did the next best thing to going back in time. He visited some volcanoes, which exhibit some peculiar chemical conditions that are rare on Earth now, but were more common four billion years ago.

In places such as Iceland, Hawaii, and Mount Mutnovsky in Kamchakta, Russia, volcanic eruptions heat the surrounding land to such an extent that the area is sterilized for years to come. Little ponds of hot, acidic water often form in the area, constituting an environment similar to what scientists think existed on Earth at the beginning of the Archean eon just before the emergence of life (Figure 4). On one of his trips in 2009, Deamer conducted an experiment. Into pools of hot, acidic (pH 3) volcanic water, he poured in mixtures of the kinds of fatty acids, amino acids, and other compounds present in the Murchison meteorite. Just as had happened in the lab, lipid membranes spontaneously formed, producing tiny liposomes despite the low pH. And just as they did in his 1977 laboratory experiment, building blocks such as amino acids often were enclosed within them.

Figure 4: Artist's rendering of ancient hydrothermal pools. The inset picture shows a modern analogue, Bumpass Hell in California. Image from Damer, B. & Deamer, D. (2015). Coupled phases and combinatorial selection in fluctuating hydrothermal pools. Life, 5(1): 872-887.

image ©Bruce Damer and David Deamer
Comprehension Checkpoint
Volcanoes have chemical conditions similar to those of early Earth.

Primeval membranes: Free fatty acids versus phospholipids

Though highly sensitive to pH and less efficient than phospholipids at forming membranes, free fatty acids on early Earth would have offered a distinct advantage to an emerging system of prebiotic liposomes. Genetics professor Jack Szostak of Harvard Medical School achieved celebrity status for his work conducted in the 1980s showing how telomeres protect chromosomes. The research earned him a Nobel Prize in 2009, but since the 1990s he has been working on the origins of life. Being a geneticist, Szostak started out from an RNA/DNA perspective, trying to understand how RNA could form on the early Earth from its building blocks. But like Deamer, Szostak recognized early on that primeval lipid compartments would be needed to make the whole thing happen. So he began studying fatty acids and phospholipids and the different kinds of liposomes that they could form under simulated primeval conditions.

Labeling both free fatty acids and phospholipids with special fluorescent molecules, Szostak was able to monitor and compare the physical behavior of liposomes formed from fatty acids with those formed from phospholipids. He found that both kinds of liposomes could form in a wide range of sizes, but that the liposomes made of free fatty acids were more dynamic. Their fatty acids jump around constantly, not only within the membrane of each liposome, but also between different liposomes.

Thus, liposomes are constantly exchanging fatty acids, and yet as a unit each liposome remains stable. On the pre-biotic Earth, this would allow individual liposomes to exist on a continuous basis, while “trying out” different types of membrane fatty acids. At the same time, anything inside the liposomes – RNA or DNA building blocks, for instance, and any polymers made from them – would also be exchanged between liposomes. Effectively, spontaneously forming primeval liposomes would be “protocells,” tiny laboratories for isolating and testing the stability and chemical abilities of macromolecules like RNA (Figure 5).

Figure 5: An artist's rendering of a protocell, showing the fatty acid membrane surrounding RNA ribozymes.

image ©Janet Iwasa, Szostak Laboratory, Harvard Medical School and Massachusetts General Hospital
Comprehension Checkpoint
Which would make a better primeval membrane?

Liposomes: From building blocks to polymers

The incorporation of building blocks of RNA, DNA, and proteins within spontaneously forming liposomes would not have been enough to trigger abiogenesis. To move from prebiotic chemistry to a truly living cell, a system of molecules would need to develop copying capabilities, and not just the ability to copy any molecule, but specifically to copy themselves. Such ability requires polymers because only polymers can store and manage information. This raises a question, namely how could a collection of building blocks polymerize while enclosed within a tiny compartment? RNA can have catalytic ability, and Lincoln and Joyce have shown that such catalytic ability can evolve. But the Lincoln-Joyce experiment started with RNA enzymes, not individual nucleotide building blocks.

A major insight toward unraveling this conundrum came when Szostak started working with Montmorillonite clays. These clays are created when volcanic ash is subjected to weathering. Researchers think they were present on early Earth because of high levels of volcanic activity during that era. Montmorillonite clays are capable of facilitating chemical reactions on RNA nucleotides – indeed on many different types of biological building blocks. For this reason, Szostak began working with Montmorillonite both with RNA/DNA nucleotides and fatty acids. He found that not only do the clays catalyze nucleic acid polymerization, but they also help bring fatty acids together to form the lipid bilayers. In other words, the same entity that helps form membranes also helps RNA form repeating molecules, called oligomers. Oligomers are structurally similar to polymers, but are smaller in size.

On top of this, both Szostak and Deamer found in slightly different ways that lipids themselves can help RNA nucleotides link up to form RNA polymers. The process is simple: fatty acids are placed on a microscope slide and allowed to dry out. Then, RNA nucleotides are added and the slide is dried out again. Then water is added and the slide is dried out yet again. As these steps are repeated in succession, gradually, nucleotides link up to form strands of RNA. The linking happens during the drying in a reaction called dehydration synthesis, and the shape of the fatty acid molecules helps the reaction occur.

The fact that both fatty acids and Montmorillonite clay can assist the formation of long RNA molecules shows that there are different possible pathways to the same result, which is a good thing in origins of life research. In order to go from molecules swirling in a soup to a living cell, many chance events are necessary, so the more ways that something is possible, the more likely it is to occur.

Furthermore, this result also argues that, once RNA nucleotides formed and became enclosed within a primitive membrane, this would not only have concentrated the nucleotides, but also helped them unite into RNA molecules (Figure 6) that could then evolve into enzymes and/or store information. If this happened over and over, the RNA molecules would get longer and longer and recombination of different sections would occur, as occurred in the Lincoln–Joyce experiment. Effectively, the primeval liposomes would be natural laboratories for a wide variety of creative RNA chemistry.

Figure 6: A single strand of ribonucleic acid (RNA) showing the four bases: adenine (A), cytosine (C), guanine (G), and uracil (U).

Comprehension Checkpoint
During a reaction called dehydration synthesis, nucleotides link up to form

Compartments for nature’s tinkering

In prebiotic times, the lipid membrane-bound liposomes that formed spontaneously and enclosed biological building blocks and catalyzed their polymerization would have allowed nature to run multiple chemical “experiments.” When we talk about a “test tube,” for instance in connection with the Lincoln-Joyce experiment, we don’t really mean just one glass container. Usually biologists run multiple samples side by side in test tube-like wells on plates containing multiple wells. This allows for multiple copies of the same kind of sample and also for different conditions and mixtures that can be compared. When working manually, biochemists commonly use 96-well plates.

In certain areas of research, particularly in the pharmaceutical industry, robotic technologies are used to run thousands or hundreds of thousands of differing reactions side-by-side. The lipid-bound liposomes are nature’s way of doing the same thing, but with a scale no pharmaceutical company could ever match. Over the course of hundreds of millions of years, the entire world was like a giant laboratory with trillions, even quadrillions of liposomes acting as tiny test tubes. When the numbers of “trials” is that high, even extremely rare and improbable events are sure to happen at least occasionally. You might think that shuffling a deck of cards and ending up with all four aces on top would be impossible. It’s not impossible; it’s improbable. If you try thousands of times, it will eventually happen.

So it was with early Earth. Trillions of liposomes were forming, growing, splitting, and capturing inside them whatever molecules happen to be nearby. Once formed, the liposomes were helping the captured building blocks to polymerize. Since the liposomes were dynamic, exchanging fatty acids between their membranes, along with various molecules from within their interiors, each liposome had a chance of capturing something novel and add it to its interior. If a liposome then captured an RNA- or protein-based enzyme that helped the lipid metabolism even further by converting the energy of sugars into the formation of lipids, that would be a further enhancement. It would allow the construction of more membrane material that could separate into a daughter liposome. Such liposomes would be protocells at that point. Not life, but well on the pathway to it.

Any protocell with an RNA enzyme that could make crude copies of itself, either acquired from outside, or built inside, would accumulate multiple copies of that RNA enzyme. The copies would recombine like the RNAs in the Lincoln-Joyce experiment and Darwinian selection would be in full swing. Selection would favor molecules, or systems of molecules, that could self-copy with ever increasing accuracy and efficiency.

If all of this happened in one protocell, it would also happen in many. And so, Darwinian selection would expand from the molecular level to the protocell level. Those protocells with the best copying molecules inside, and also with the best helping molecules, would have a competitive advantage. Gradually, the sieve of natural selection would favor protocells with ever-increasing abilities that could help the process of reproducing the self-replicating molecules. That would mean reproducing the entire system within the protocell's membranes.

By reproducing the entire system within such a protocell, by building new membranes to enclose new copies of the entire system, effectively, the protocells would be reproducing just like tiny organisms. In such a system, chemical reactions and the molecules needed to catalyze them would all depend on one another. Unlike the earliest liposomes containing mere building blocks, evolution at the protocell level would work in favor of everything with the protocell membrane. Complexity would increase incrementally to improve the reproductive capabilities and all protocell functions needed to support those capabilities. Gradually, imperceptibly, the first living cells would have emerged.

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