Astrobiology in the Folds

The traditional 2° structure of the 23S rRNA of E. coli. This structure is based on phylogenetic data, and is shown as two fragments. The structure contains six domains (Domain I, purple; Domain II, blue; Domain III, magenta; Domain IV, yellow; Domain V, pink; Domain VI, green). Credit: Petrov et al. (2013)

Astrobiology is often associated with the search for life on planets like Mars, or the hunt for Earth-like worlds around distant stars. However, a great deal of the research that goes into understanding life’s potential in the Universe takes place much closer to home.

For now, Earth is our only example of a habitable planet, and before we can identify life as we know it among the stars, we first have to figure out exactly what we’re looking for.

This is why many astrobiologists are busy studying the fundamental ways in which living cells function, evolve and survive. Their work is an essential step in defining how life began on our planet, and in locating other places in the Universe where life could gain a foothold. Sometimes, searching for life in the vast reaches of space begins by looking inward to the microscopic complexity all around (and inside) of us.

The Structure of Ribosomal RNA

Astrobiologists supported by the NASA Astrobiology Institute (NAI) have revealed new information about the structure of RNA molecules found in the ribosomes of cells. Ribosomes are an essential cog in the machinery of a living organism. They play a vital role in building proteins in every cell of every organism on Earth.

Studying the three dimensional structure of ribosomes helps scientists understand how they work, the role they play in living cells, and what they can teach us about the evolutionary history of life as we know it. The problem is, the structure of these assemblies is very complicated. They are composed of proteins and strands of RNA that twist, turn, loop and fold around one another in complex, but specific patterns.

Scientists made a big step in untangling this mess when they determined the secondary structures (2° structures) of RNA segments that help make up a ribosome. Today, scientists have defined hundreds of 2° structures for ribosomal RNAs (rRNAs), and from all kinds of organisms, but the first rRNA 2° structures came from bacteria. These structures were established in 1981 as the basic framework for all 23S rRNA 2° structures.

The 23S rRNA is now an iconic example of 2° structure, and you can find it in nearly all biology and biochemistry textbooks.

The new 2° structure based on data from high-resolution 3-D models (from E. coli). This structure accurately represents all helices, and contains seven domains (Domain 0 in orange; Domains I-VI are colored as in panel 1a). In the traditional 2° structure shown above, a central single-stranded region of rRNA is partitioned between multiple domains. In the new 2° structure, that same rRNA is double-helical and is fully contained within Domain 0. Credit: Petrov et al. (2013)

2° Structures represent folding elements found in rRNA (the loops, base pairs, stems, single strands, etc.), and they help scientists visualize how a strand of rRNA folds. Studying this folding provides a great deal of information about how the rRNA was assembled, how it has evolved, and how it functions in living cells.

In 2001, scientists made another big step forward when they used X-ray crystallography to build high-resolution 3-D structures of ribosomes. Now, we have 3-D structures from organisms in all three domains of life (Archaea, Bacteria and Eukaryota). These high-res models helped validate the previously-defined 2° structures for 23S rRNA and the methods used to build it.

However, the 3-D structures also showed that the traditional 2° structure was not perfect. Both the 3-D and 2° models had significant sections at the center of the molecule that were difficult to interpret. This is a big problem, because inaccuracy can lead to incorrect interpretations of 23S rRNA folding and function.

But how could scientists have had it wrong for so many years?

“It speaks to how complicated the ribosome is, and how difficult it is to understand on a molecular level,” says Dr. Loren Williams, Principle Investigator for the NAI team at Georgia Tech and co-author of the paper. “We worked with it for years without realizing it was wrong.”

Wrestling with Ribosomes

Williams and his team, including Anton Petrov and Chad Bernier at GA Tech’s Center for Ribosomal Origins and Evolution, used data from the high-resolution 3D structures to ‘re-build’ the 2° structure of 23S rRNA, as well as a other rRNAs known as the 5S and 16S rRNAs. Their hope was to improve the accuracy of 2° structures in order to make them consistent with the 3D structures. Such a feat would greatly improve the usefulness of 2° structures for scientific studies.

“Over the years we have worked hard to develop tools that allow us to understand the ribosome in a fundamental molecular way, and to quickly map diverse types information onto the ribosome,” said Williams. “We have automated a substantial part of the process. The automation, and even the process of setting up the automation, forced us to the realization that the traditional 2° structure was wrong.”

23S rRNA is found in all living cells, but there are slight differences in molecules taken from different species. Studying the similarities and differences provides important clues about how the molecule has evolved throughout the history of life. The researchers used data from as many 23S rRNA molecules as possible. This included information from 122 organisms, and represented all three branches on the tree of life (19 eukaryotic species, 67 bacterial species and 36 archaeal species).

This image is a space filling representation of Domain 0 from 23S rRNA of E. coli. Credit: Petrov et al. (2013)

Ultimately, the team created new 2° structures based on phylogeny and 3-D structures, and found that they were more accurate than previous 2° structure that were based on phylogenetic data alone. Their new structures highlighted significant changes that need to be made in the accepted 2° structure of 23S rRNA.

Previously, it was thought that 23S rRNA consisted of six pieces (called ‘domains’) attached to a central core. Most of the problems with the old 2° structure of 23S rRNA are found in this central core. While the six domains were carefully mapped, the core was like a elongated string. The 3-D structures that came later seemed like messy balls of string.

“I remember when the first X-ray crystal structure of the large ribosomal subunit was published,” says Professor Jonathan Dinman, of the Department of Cell Biology and Molecular Genetics at the University of Maryland. “They made this point of saying that, while the domain structure in the rRNA of the small subunit is really apparent, the large subunit is folded up like crazy origami and you can’t make sense of. That view was reinforced by the old 2-D structure map.”

The new 2° structure outlined by Williams and his team at Georgia Tech makes some major changes.

“I saw their map and I said ‘WOW this is really cool,’” continued Dinman. “In the old 2° structure maps of the large subunit, everything is sort of disjointed and hanging out in space on its own. In this 2-D structure map, they’ve closed the holes. They’ve closed the gaps, and you can begin to see how this thing folds up on its own.”

The new map reveals that the central core of 23S rRNA, which was previously too difficult to interpret, is actually a separate domain. 23S rRNA contains seven domains instead of the traditional six, and this has some major implications in understanding how the rRNA actually folds and functions.

Ancient Machinery of Life

This isn’t the first time that the 2° structure of rRNA has been refined since its initial discovery in 1981. As methods for studying rRNA have grown more sophisticated, the structural models are providing increasingly detailed information about the evolution of life on Earth.

Such small-scale studies of how life works at the cellular and molecular level may seem far removed from the search for life’s potential on other worlds in the vastness of space – but studying biological and chemical mechanisms in cells is essential in understanding what actually constitutes ‘life.’

Anton Petrov (Left), Loren Williams (Center) and Chade Bernier (Right) of Georgia Tech. Credit: Loren Williams/GA Tech

Studies like those conducted in Loren William’s lab provide essential clues about the requirements for life’s origin and evolution, and the types of molecules that might provide evidence for life as we know it on distant planets and moons.

“The ribosome is older than life itself,” explains Dinman. “We think about origins of life, and the ribosome is at the center of it all. The ribosome is what really is the mediator between the world of nucleic acids and the world of amino acids. This is ancient machinery that predates cellular life.”

At some point in Earth’s history, pieces of RNA were gathered around a central core. Somehow, this gave early life the ability to use both nucleic acids and amino acids, and to translate RNA into protein. Understanding how this critical step in life’s history happened could advance many areas related to the study of life’s origins.

“We’re going to use this map in our subsequent publications,” comments Dinman. “I think this is a tremendous tool and I would encourage other people in the field to use this map and move forward.”

The Georgia Tech research team has released high-resolution, editable versions of their results online in the hope that other researchers can evaluate and add to the revisions they have made (http://apollo.chemistry.gatech.edu/RibosomeGallery).

“The gallery web site is our attempt to make our results available to everyone,” says Williams. “In the ‘read-me’ file, you can see that we provide a lot of secondary structures. We provide the files in formats that are easy to manipulate and we give license to do that. We are trying to make the ribosome available to the people.”

The paper, “Secondary structure and domain architecture of the 23S and 5S rRNAs,” was published in the journal Nucleic Acids Research in June, 2013, and is also freely available online: http://nar.oxfordjournals.org/content/early/2013/06/14/nar.gkt513.full#xref-ref-43-1