In the quest to understand the origins of life on Earth, scientists are finding fresh evidence that bundles of RNA called ribozymes were the first truly biological molecules.

In the quest to understand the origins of life on Earth, scientists are finding fresh evidence that bundles of RNA called ribozymes were the first truly biological molecules.

In a curtained-off corner of his windowless laboratory, David Lilley is peering at the molecular machinery that may have helped life get its first foothold on our planet. Illuminated only by a laser beam bouncing off a series of carefully positioned prisms and mirrors, Lilley - who heads the Cancer Research UK Nucleic acid structure research group at the University of Dundee, Scotland - is studying individual ribozymes in action. 

These strands of ribonucleic acid (RNA) are capable of catalysing rudimentary chemical reactions, and since their discovery in 1982 many scientists have argued that this kind of molecule probably played a key role in the origin of life. 


Source: © David Lilley, University of Dundee

David Lilley’s colleague Carlos Penedo watches his ribozymes do their stuff

’In my more fanciful moments, I like to think we’re glimpsing what a world 3.6 billion years ago must have been like,’ says Lilley. If scientists can pin down exactly how ribozymes work, it may help to uncover how proteins, DNA and ultimately the first living creatures sprang into existence, and resolve an apparent paradox at the heart of genetics.  

Enter the RNA world 

In almost every cell of our body, entwined strands of DNA hold the genetic instructions for making worker molecules such as proteins. DNA provides a template for the synthesis of RNA, which carries the genetic code into the jelly-like cytoplasm that fills a cell. Once there, its chemical data is used to construct proteins, some of which are the enzymes that speed up biological reactions. 

But this simple story ignores the fact that DNA replication and RNA synthesis themselves rely on enzymes. And since enzymes cannot form without DNA, and DNA cannot form without enzymes, the paradigm posed a knotty chicken-and-egg problem for biologists. 

’Nobody could imagine how you could evolve a system from scratch that involved DNA being transcribed into RNA and then some sort of protein translation machinery,’ says Lilley. ’That can’t come out of nowhere.’  

The answer for many scientists is in the RNA world. This hypothesis suggests that the earliest self-replicating precellular lifeforms had neither DNA nor proteins, but relied instead on RNA enzymes - ribozymes - to act as both code carrier and catalyst for primitive biochemistry (see box, p45). 

Ribozymes are widespread in nature, and some play a fundamental role in the everyday business of the cell. For example, ’riboswitches’ are ribozymes found in many bacteria, which when bound to specific target molecules can control the formation of messenger RNA and the proteins they help to build, without needing any assistance from regulatory proteins. In addition, scientists recently found that a ribozyme lies at the heart of one of the most important biochemical reactions of all - the formation of peptide bonds between amino acids to build proteins.  

This takes place in the ribosome, a protein factory found in all living cells that is made from a few strands of RNA and a rabble of proteins. When crystallographers solved the three-dimensional structure at the heart of this machine in 2000, it was immediately obvious that RNA strands, and not protein molecules, were responsible for forming the peptide bonds. 

’There’s no sign of amino acids around to help the catalysis,’ says Michael Gait, a chemist at the Medical Research Council’s Laboratory of Molecular Biology at the University of Cambridge, UK. ’So the primitive ribosome may well have been made up of mostly RNA.’  

Protein-based enzymes generally make far better catalysts than RNA-based ribozymes, so once protein production was up and running in the ancestral ribosome it’s possible that most ribozymes would have become redundant. 

This suggests that the RNA world could have been populated by a much wider spectrum of ribozymes with a more spectacular array of talents. To prove the point, researchers have spent the past decade creating ribozymes by getting them to evolve in the test tube and then screening for particular biological activities. This approach has thrown up many novel ribozymes, says Gerald Joyce of the Scripps Research Institute in La Jolla, California, US, including some that help to create the very nucleotides that make up RNA molecules themselves. 

Although these synthetic ribozymes make pretty crude enzymes, the work demonstrates that it is at least possible for RNA to do the basic jobs needed for simple metabolism. ’It’s not so ridiculous to imagine that primitive cells - some fatty bag of RNA and a few other chemicals - could have replicated,’ says Lilley. And once replication is under way, evolution by natural selection can do the rest, he says. 

Tricks of the ribozyme trade 

RNA is a simple molecule, at least when placed alongside a protein. It comprises a backbone of alternating ribose (a five-carbon cyclic sugar) and phosphate, with each attached to a ’nucleobase’, most often one of adenine, cytosine, guanine or uracil. These four bases have rather similar properties. ’They have a few functional groups like carbonyl groups and exocyclic amines,’ says Lilley, ’but that’s it.’ Proteins, by contrast, are nature’s chemistry set. Their amino acid ingredients have side chains that are ready for just about every aqueous reaction you can think of, he says.  

So how is it that humble ribozymes are able to pull off so much interesting chemistry? It seems likely that many ribozymes on the early Earth could have operated in concert with the resources around them. One tactic is for RNA’s negatively-charged phosphate groups to fold in conjunction with metal ions. The three-dimensional structure of a folded strand of RNA is therefore influenced by the metal, which can participate directly in the catalytic chemistry. Not only can they stabilise a reaction’s transition state, they also bring water molecules into the equation, which could potentially be involved in acid-base catalysis.  


Source: © David Lilley, University of Dundee

The structure of the VS ribozyme has revealed its catalytic mechanism

Some ribozymes, however, are capable of intricate chemistry all on their own. The hairpin ribozyme can bring two loops of RNA close to each other, carefully cutting and pasting them together about a million times faster than would normally occur. Scientists believe that a specific guanine nucleobase within the complex helps to drive this acid-base reaction. 

Lilley has recently tested this hypothesis by synthesising a completely new hairpin with the suspected guanine nucleobase replaced by an imidazole group. Both guanine and imidazole can act as bases, but they work best at different pHs. While the imidazole-substituted ribozyme catalyses RNA chemistry just as well as its natural analogue, the change in the pH-dependence of the reaction confirms that the original guanine nucleobase was a crucial reaction centre in the original hairpin, says Lilley. 

Seeing is believing 

This detailed understanding of ribozyme activity is just one example of what the network of laser beams in Lilley’s darkened laboratory can deliver. ’We can actually watch the chemistry occurring almost before our very eyes,’ says Lilley.  

He uses a technique called fluorescent resonance energy transfer (Fret), which involves tagging ribozymes with a couple of fluorophores - fluorescent molecules that each absorb light at one wavelength and re-emit it at another. If one fluorophore emits light at a wavelength that its partner can absorb, energy is transferred between them. The closer the two fluorophores are to each other, the more efficient the process, so measuring the energy transfer can reveal the precise location of the two groups. 

Until recently, it has only been possible to study the energy emissions from an ensemble of large molecules. ’I’ve spent my whole career trying to deduce the properties of molecules when I’m looking at about 1015 of them,’ says Lilley. Now CCD (charge coupled device) cameras and ’avalanche photodiodes’ can achieve sufficient resolution to home in on a single ribozyme going about its business. ’Suddenly you can see one molecule oscillating between two or more states,’ he says. ’You know where you came from and you know where you go to next - that’s a remarkable thing that you’ve never had in kinetics before.’ This kind of insight is not cheap: one of the CCD cameras in Lilley’s inner sanctum set him back some ?30,000. 

Deeper origins 

While ribozymes may have been the chemical catalysts of early life, a deeper question remains: where did the ribozymes themselves come from? 

For more than half a century, scientists have proposed that simple chemical precursors found on the primitive Earth could have formed the essential components of biological molecules such as RNA. The famous Miller-Urey experiment of 1953 showed that amino acids could form in a mixture of methane, ammonia, hydrogen and water that was sparked with arcs of electricity. 


Source: © Nasa

Artist’s impression of IRS-46 and its dust cloud

But Earth-bound synthesis of life’s building blocks would have been tricky during the first billion years of the planet’s life, which saw intense volcanic activity and volleys of massive meteorites raining down on the surface. 

’Yet somehow life emerged relatively quickly,’ says Nigel Mason, professor of physics and astronomy at the Open University in Milton Keynes, UK. It may be possible that the ingredients to make RNA were already present in space and hitched a lift to Earth on a meteorite or comet, he says. 

Recent astrobiology research supports this idea. In 2005, Nasa scientists claimed to have found the ingredients of one of the four nucleotide bases drifting in a distant solar system. They used the Spitzer Space Telescope - the largest infrared telescope ever launched into space - to study dust clouds surrounding 100 young Sun-like stars in the Milky Way. Similar dust clouds are thought to have spawned our own planet more than four billion years ago.  

Spitzer found both acetylene and hydrogen cyanide in the dust surrounding one young star called IRS 46, which lies some 375 light years from Earth. The dust is in a temperate region where water could exist as a liquid, dubbed by optimistic astrobiologists as ’the habitable zone’. 

’If you add hydrogen cyanide, acetylene and water together in a test tube and give them an appropriate surface on which to be concentrated and react, you’ll get a slew of organic compounds,’ says Geoffrey Blake of the California Institute of Technology, Pasadena, a co-author of a paper announcing the discovery in Astrophysical Journal Letters  in January this year. One of these compounds is adenine. 

Although adenine itself has not yet been spotted in the dust cloud, ’at least we now have the ingredients,’ says Mason. Similar high-tech inspections of space dust have turned up evidence of other molecules, such as formic acid and acetic acid. ’In the region where planets form, the chemistry is much more complex than we thought,’ he says. 

But while the ingredients of RNA could hitch a ride to Earth on a meteorite, it is still a huge step from these to a self-replicating molecule, cautions Martin Line, a microbiologist at the University of Tasmania, Australia. 

’The real problem is their very low concentration,’ he says. Perhaps the components of the first RNA molecule were dissolved in a small lake that slowly evaporated, concentrating the compounds until they began to link up, he speculates. Add amino acids to the mix, and the soup could have been just a few reactions away from the very first proteins.  

With ribozymes already strong contenders as the catalysts of creation, Lilley says that the next big challenge is to figure out exactly how their RNA world was born from just a few simple ingredients. 

Henry Nicholls is a freelance journalist based in London, UK

En route to an RNA world 

In a prescient article on the origin of life published in American Naturalist  in 1917, Harvard psychologist Leonard Troland postulated a primitive molecule - what he called a ’genetic enzyme’ - capable of catalysing reactions of both itself and other molecules. This makes Troland’s hypothetical molecule an early forerunner of the ribozyme, says Iris Fry, a historian of science at Tel Aviv University in Israel and author of The Emergence of Life on Earth. 

It took until the 1960s, when it became clear that RNA had a hand in many aspects of cell function, for scientists to predict that some RNAs might be capable of catalysis. Carl Woese of the University of Illinois, Francis Crick at the UK’s Medical Research Council and Lesley Orgel of the Salk Institute in San Diego independently proposed that this dual-purpose molecule could unlock the secret of life without DNA or proteins. It was only in the early 1980s that empirical evidence for ribozymes finally emerged. Thomas Cech, of the University of Colorado, US, managed to show that RNA splicing in the ciliated protozoan Tetrahymena thermophila  was being catalysed by RNA rather than a protein enzyme. Cech shared the 1989 Nobel prize for chemistry with Sidney Altman of Yale University for the discovery of these multi-talented molecules. In 1986, Harvard University professor Walter Gilbert coined the phrase ’RNA world’ in an article in Nature.