[The birth of this molecule that links amino acids into chains
would have created a fundamental shift in the the prebiotic world,
providing a key ingredient to all life as we know it. Scientists are
learning to build it.]
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HOW DID LIFE BEGIN? ONE KEY INGREDIENT IS COMING INTO VIEW  
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Amber Dance
February 28, 2023
Nature [[link removed]]

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_ The birth of this molecule that links amino acids into chains would
have created a fundamental shift in the the prebiotic world, providing
a key ingredient to all life as we know it. Scientists are learning to
build it. _

,

 

Billions of years ago, before there were beasts, bacteria or any
living organism, there were RNAs. These molecules were probably
swirling around with amino acids and other rudimentary biomolecules,
merging and diverging, on an otherwise lifeless crucible of a planet.

Then, somehow, something special emerged: a simple machine, a pocket
made of RNAs, with the ability to place amino acids next to one
another and maybe link them into chains. This was the macromolecule
that would gradually evolve into the ribosome, the RNA–protein
complex responsible for translating genetic information into proteins.
Its birth — the details of which remain hypothetical — would have
created a fundamental shift in this prebiotic, RNA-dominated world,
providing a key ingredient to all life as we know it. Ada Yonath, a
structural biologist at the Weizmann Institute of Science in Rehovot,
Israel, and her team first conceptualized this ‘protoribosome’
idea nearly two decades ago, after she and others determined the
structure of the modern ribosome, a feat that later secured Yonath a
share of the 2009 Nobel Prize in Chemistry.

But to solidify the case for the hypothetical protoribosome, Yonath
and her laboratory would have to build it.

It’s a project that other scientists have watched with interest. And
the lab’s achievements in the past two years — creating a
primitive RNA machine that can link two amino acids together1
[[link removed]],2
[[link removed]] — have
created a ripple of excitement. A group in Japan, working separately
and led by molecular biologist Koji Tamura at the Tokyo University of
Science, has succeeded in creating a similar, functional
protoribosome3
[[link removed]].
“It’s strongly supportive of Ada’s protoribosome idea,” says
Paul Schimmel, a biochemist at Scripps Research in La Jolla,
California. “I’ve always had an open mind; the open mind is
becoming a more convinced mind.”

Although doubts and caveats remain, Yonath’s and Tamura’s work
seems to recapitulate a milestone on the road from primordial organic
molecules to the ribosome used by the last common ancestor of all
living things. This was no simple task: In Yonath’s group, the
project was passed from researcher to researcher and took more than 15
years to succeed. The work has now opened the door for origin-of-life
scientists to fill in further details. And others are looking at the
protoribosome, or something like it, as a tool to create new kinds of
biomolecule.

“This should be a starting point of many more fields of research,”
says Tanaya Bose, a postdoctoral researcher who has led the Yonath
group’s efforts for the past several years.

CRYSTAL INSPIRATION

Scientists have been attempting to recreate some semblance of the
chemical origin of biomolecules for decades; it was 70 years ago that
Stanley Miller, a chemist at the University of Chicago in Illinois,
sparked a gas mixture to create organic compounds4
[[link removed]].
Researchers including Carl Woese and Francis Crick suggested the
ribosome could have started as a molecule made solely of RNA, an idea
that was supported 40 years ago with the proof that RNAs can catalyse
reactions5
[[link removed]],6
[[link removed]]. This led
to the ‘RNA world’ hypothesis, which describes a time, before
cells or actual life, when RNAs replicated and catalysed reactions7
[[link removed]]. The
RNA-world idea has come into doubt in the past decade; many scientists
now suspect that a variety of biomolecules, including rudimentary
proteins, lipids and metabolites, might have existed together with
nucleic acids.

Yonath suspects there were plenty of RNAs on early Earth. “Most of
them don’t exist any more because they were small and not useful.”
The protoribosome, she says, was the one that lasted.

Yonath and her colleagues published high-resolution structures for the
two protein–RNA subunits that make up a ribosome in the early 2000s8
[[link removed]],9
[[link removed]]. These
examples came from extremophile bacteria. And as ribosomal structures
from other organisms were published, Ilana Agmon, a scientific adviser
in Yonath’s group, noticed something that she found striking. Deep
in the core of the large subunit was a semi-symmetrical segment. This
region contained a pocket-like structure, made of ribosomal RNA,
called the peptidyl transferase centre (PTC). During the translation
of mRNA into protein, when two amino acids are placed in the PTC, it
creates conditions for them to click together. And, although the
specific nucleotide sequence of the structure varied between species,
the shape was the same in every example, suggesting that it’s
crucial to the ribosome’s ability to support life (see ‘The heart
of the matter’).

[The heart of the matter: a graphic that shows the location of the
PTC, an RNA structure that is highly conserved, in a ribsome..]

Source: refs 1 & 3/PDB.

In 2006, when Yonath started asking her team about the evolution of
the ribosome, Agmon suggested looking closely at the semi-symmetrical
region containing the PTC. “We speculated that this is the
protoribosome, this is the part from which the ribosome evolved,”
recalls Anat Bashan, a senior staff scientist who’s been involved in
the project since the beginning. But, the region they were positing as
the putative protoribosome is assembled from 178 ribonucleotides,
which Yonath noted would be an awfully large structure to spring into
being, fully formed, on primordial Earth. Based in part on the
symmetry she’d observed, Agmon proposed a model requiring 2 similar,
L-shaped RNAs of 60 and 61 nucleotides. The team thought this was a
more reasonable size of molecule to arise on early Earth.

Fragments of that size are certainly plausible. Elisa Biondi, a
biochemist at the Foundation for Applied Molecular Evolution in
Alachua, Florida, and her colleagues managed to synthesize RNAs of
about 100–300 bases long on materials called rock glasses, which
would have formed owing to volcanic action or meteor impacts in a
prebiotic world10
[[link removed]].

But not everyone is convinced that Yonath’s and Agmon’s short
fragments arose spontaneously. Joseph Moran, an organic chemist at the
University of Strasbourg in France, praises Yonath’s accomplishments
but doubts that the protoribosome just popped into existence. “It
had to be derived from much simpler things,” he says.

Robert Root-Bernstein, a biologist at Michigan State University in
East Lansing, has a theory for what those simpler things might have
been: transfer RNAs (tRNAs). In the modern ribosome, amino acids enter
the PTC attached to tRNAs that match the three-letter codes on the
mRNA. Those codes determine which amino acid comes next in the
protein. To Root-Bernstein and others, the PTC core looks a lot like
four tRNAs joined together. And tRNAs, he notes, don’t just deliver
amino acids to the ribosome; they are versatile molecules that can do
all kinds of task, such as sensing nutrients and silencing genes.
Perhaps they had some function before the protoribosome, then provided
the building blocks for that structure.

EXTRAORDINARY EVIDENCE

Regardless of how the hypothetical protoribosome came to be, at the
time when Agmon and Yonath first conceived it, there was no
experimental evidence to show that it could have existed and worked as
they thought it did. The protoribosome hypothesis assumes that one of
those early RNA pockets could link amino acids together, and that it
then evolved into the ribosome, which has that same function.
Scientists who spoke with _Nature_ say that’s a fair idea, but
it’s not a given. “It’s plausible,” says Anton Petrov, an
evolutionary biologist at the Georgia Institute of Technology in
Atlanta, but he also thinks that early RNA machines might have had
functions distinct from peptide synthesis, and that they then took on
that role later as the protoribosome emerged.

“It was a very bold and imaginative suggestion from Yonath,” says
John Sutherland, a chemist at the Medical Research Council Laboratory
of Molecular Biology in Cambridge, UK.

[[link removed]]

Oldest-ever DNA shows mastodons roamed Greenland 2 million years ago
[[link removed]]

Bold theories require extraordinary evidence, and that’s what the
lab set out to obtain. After developing the protoribosome hypothesis,
Agmon moved to the Technion — Israel Institute of Technology in
Haifa. Graduate student Chen Davidovich took the lead as the
experiments commenced. The first step, Yonath says, was to produce the
molecules to build this theoretical protoribosome.

Davidovich studied the RNA sequences of various modern ribosomes. The
macromolecule contains dozens of ribonucleotides and accessory
proteins, but not all of these are involved in the PTC’s shape or
functions. He stripped out whatever seemed like it would be extraneous
to the protoribosome, leaving just enough RNA to create that
semi-symmetrical pocket2
[[link removed]]. Some of
these RNAs were able to pair up into something similar to the PTC core
that Agmon had imagined.

“This took a long time,” Yonath recalls — but the next step
would take even longer. The second step was to show that these
putative protoribosomes could take two amino acids and hook them
together.

Davidovich attempted to show PTC activity in the same way that other
researchers have measured the ability of the modern ribosome to link
two amino acid analogues into a dipeptide. He labelled such analogues,
linked to a handful of nucleotides to stand in for tRNA, with
radioactivity. After mixing them with the protoribosome, he thought he
would be able to sort the molecules by size and find longer,
radio-labelled dipeptides. But he never saw a hint of dipeptide. “It
almost killed my career,” recalls Davidovich. Fortunately, he had
back-up projects related to another interest of Yonath’s lab,
antibiotics that attack ribosomes. He graduated in 2010 and now leads
a lab studying gene repression at Monash University in Melbourne,
Australia.

Next up was graduate student Miri Krupkin, who was drawn in by the
symmetry of the PTC and its ability to build proteins quicker than any
human chemist has managed. Using Davidovich’s constructs and others
that Krupkin designed herself, she too attempted to detect the
radioactive dipeptide product on the basis of on its size. She also
tried labelling the substrates with fluorescence, instead of
radioactivity. Still, nothing.

Krupkin began to wonder if the protoribosome even made peptides when
it first emerged. She tested other potential chemistries, such as
adding or removing phosphate groups from other molecules, and still
came up dry.

[[link removed]]

Origin of life theory involving RNA–protein hybrid gets new support
[[link removed]]

“I kept going, because it’s such an interesting question,” says
Krupkin. But similar to Davidovich, she had to rely on another
project, also on antibiotics, to graduate in 2016. She’s currently
investigating HIV’s RNA structure as a postdoc research fellow at
Stanford University in California.

Bose, who joined the lab as a postdoc in 2016, would take the project
to the finish line. She had trained as a chemist, and that gave her a
fresh perspective. She knew that if the primitive protoribosome worked
at all, it would probably be inefficient, yielding a minute amount of
dipeptide. Rather than separating the reaction products by size, Bose
turned to mass spectrometry, which had advanced to become the most
sensitive method by this time. It still wasn’t easy — she
performed many reactions and control experiments, and tried two forms
of mass spectrometry — but at last, she saw a peak representing the
expected dipeptide1
[[link removed]],2
[[link removed]].

“The amount of product was tiny,” says Bose. She suspects
Davidovich and Krupkin might have been making it all along, but there
wasn’t enough to detect it using their methods.

Even that tiny amount was “a bit of a feat” to produce, says
Sutherland. “They’ve made progress on a really difficult
problem.”

NEXT STEPS

By the time Krupkin was trying to measure a dipeptide product, the
Yonath group wasn’t the only one on the trail of the minimal
peptide-bonding machine. Tamura, in Japan, was also inspired by the
semi-symmetrical core pocket. He recruited a group including
master’s student Mai Kawabata and undergraduate Kentaro Kawashima to
make their own protoribosome-like structure.

Their PTC facsimile, made from two 74-nucleotide RNAs, was similar to
Yonath’s, but to stand in for modern tRNAs, they used structures
called minihelices. These are about half the size of a modern tRNA,
and were therefore considerably larger than the tRNA replacements
Yonath used. Modern tRNAs are thought to have evolved from
minihelices.

Tamura’s team eventually achieved its own success, also detecting
the dipeptide with mass spectrometry3
[[link removed]]. “Mai
and Kentaro were patient and responsive, and finally, we won a big
win,” says Tamura.

Schimmel, Tamura’s former postdoc adviser, says that the minihelices
were a key addition. “He carried it to the next level, so to speak,
in that he constructed what is believed by many evolutionists to be
the primordial tRNA,” says Schimmel. Some scientists, he notes, even
suspect that minihelices evolved the ability to self-replicate,
another key step on the way to living organisms.

[[link removed]]

The shifting sands of ‘gain-of-function’ research
[[link removed]]

Tamura cautions that neither lab’s construct works exactly like the
modern PTC does. “All of our results are too simple,” he says.
“We still have a long way to go before we really understand the
evolution of the PTC and the ribosome.”

For Bashan, it’s close enough. “We think this is how the first
proteins came about,” she says.

But as other scientists note, there were probably other ways for
polypeptides to emerge on early Earth. Linking up amino acids is
“dead easy”, says Sutherland. For example, some scientists have
proposed that amino acids plus α-hydroxy acids (a group that
includes, for example, lactic acid and citric acid), could have formed
into polypeptides during cycles of cool, wet and hot, dry conditions
on early Earth — with no RNA required11
[[link removed]]. And
last year, another team used RNA bases, although not the A, C, G, and
U ones used in modern RNA codes, to put peptides together. The
researchers proposed this could have happened, without the ribosome,
in the RNA world12
[[link removed]]. The
catch, says Sutherland: “it bore no resemblance to the way that
nature now makes peptides from RNA”.

What is special about the protoribosome work, say Sutherland and
Yonath, is that it is possible to imagine how this primitive core,
over millennia, might have accumulated extra pieces of RNA and protein
to create the modern ribosome.

Petrov and his colleagues have predicted exactly that kind of timeline
by working backwards from today’s ribosome. They analysed ribosomes
for atomic-level ‘fingerprints’ that were left in the 3D ribosome
structure when new branches of RNA were added13
[[link removed]]. This
led to a model that showed early structures matching those of
Yonath’s protoribosome, with extra pieces of RNA slowly incorporated
over time (see ‘Molecular evolution’).

[Molecular evolution: a graphic that shows how the RNA structure of
the ribosome has evolved in 6 phases over time.]

Source: ref. 13.

One obvious next step for Yonath’s team is to try to make a diverse
set of peptides that are longer than two amino acids. Bose is working
on this, but it will require different starting materials.

There is one group that claims to have synthesized a longer peptide
from a protoribosome-like structure. Yuhong Wang, a biophysicist at
the University of Houston in Texas, and her colleague reported mass
spectrometry results from experiments with a larger version of the PTC
that indicate the production of a chain of nine lysines14
[[link removed]]. Wang
thinks that there might be lysine polymers of other lengths as well.
“I think we have strong evidence,” she says, but she admits that
the controls are incomplete, and she cannot explain why strings of
nine amino acids would be the most prominent product.

Wang and others are interested in using such stripped-down ribosomes
to manufacture new kinds of biomolecule, not limited to the usual 20
amino acids or even to amino acids at all, that could have uses in
medicine or industry. For example, they might build molecules out of
amino acids that are right-handed in structure, unlike the left-handed
ones seen in life on Earth. This kind of macromolecule synthesis could
be cheaper and more environmentally friendly than other means, Wang
suggests.

Meanwhile, there’s plenty of research left to do for origin-of-life
studies. Scientists need to work out how RNAs gained the ability to
self-replicate. And they need to discover how an early ribosome would
have managed to create specific peptides encoded by primitive mRNAs.
Those processes, plus the ability to synthesize peptides, would
provide the raw materials for evolution.

There’s one more factor, Sutherland says: the early peptides made by
the protoribosome must have been somehow useful, or there would be no
evolutionary advantage to the machine’s continued existence. He
suggests a couple of speculative functions: perhaps the peptides
sequestered metal ions that would otherwise destroy RNAs. Or, they
might have helped to form early biomolecular compartments to
concentrate RNA and peptides together.

“When you get something that evolution can act on,” says
Sutherland, “the rest is history.”

_Nature_ 615, 22-25 (2023)

_doi: [link removed]

_Amber Dance [[link removed]] is a
freelance science journalist in the Los Angeles area._

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