A pioneering view of the internal structure of RNA captured by scientists at Johns Hopkins became public May 13 in a report published by the journal Science.
The work of an interdisciplinary team of chemists and biophysicists marks the first actual atomic level resolution of a protein-RNA complex from the ribosome ever to be produced and only the third RNA ever to have its complex higher-order construction revealed.
"I've been traveling around the last few weeks giving talks about what we've discovered, and people's jaws just drop to the floor," said chemistry professor David Draper, out of whose lab the work originated. "So little has been known about how RNAs fit together and what kinds of structural features keep it intact. Well, now we can actually see it in 3-D, living color."
The detailed structure is significant because so little is known about it. Also, RNA's stature has risen extraordinarily in the past two decades with discoveries that RNA not only carries the genetic blueprint to the ribosomes for protein encoding but also serves as the catalyst that carries out the reactions and does the major work of the ribosome. Its ability to both store genetic information and catalyze biological reactions identifies it as central to the origins of life and suggests that it is critical for the regulation of genes and pathogens.
Unfortunately, scientists still have only a primitive understanding of RNA. Its many entwined helical elements are tightly packed into an extremely complicated, globular structure, making even a simple viewing a difficult biophysical adventure.
By binding a particularly stable kind of protein commonly known as L11 to RNA synthesized in the lab, Draper's group created an RNA-protein complex that was at last robust enough to withstand the rigors of analysis--in this case, conducted by expert crystallographers in a neighboring biophysics lab. When data analysis finally produced the images, showing details of RNA building blocks neatly arranged in a compact structure, the scientists were awed. Within the intricate RNA folds, they discovered protein clearly serving as the critical linchpin.
"We knew that protein helped the RNA fold and made it a lot more stable," Draper said, "but we never thought it would be something so direct. What we saw was how the RNA is locked into place when the protein structure connects. You see the protein grabbing onto a little RNA base to keep it from popping out of its slot. It was one of those nice moments in science when everything comes together. All of a sudden, we knew a lot more about how things work."
For years, scientists have struggled to model RNA at its most basic level. But because of the vast complexity of the molecule and its unusual proclivity to flop easily out of shape, RNA has defied efforts to capture its image except for the simplest features, at the primary or secondary levels of detail.
Consequently, important questions, such as how do proteins recognize RNA and how does RNA fold, have eluded researchers. Even as scientists have begun to understand how significant RNA is to the origins of life, proper techniques for simply seeing its detailed structure have proved difficult to impossible to manage because of RNA's inherent tendency to be unstable.
The trick, Draper discovered, was to use the particularly stable protein in combination with an unusually sturdy RNA from organisms that live in hot springs. It was also important that Draper's group chose an RNA whose secondary structure had already been detected by previous scientists but which also presented solid evidence that there was substantial additional structure at the tertiary level, the next significant level of complexity.
"We were convinced that the RNA folded in some compact, stable way, but figuring out how it did that was just impossible," Draper said. "We tried to create models, but there was simply not enough known about the rules for how RNA folds to do anything reliable. I talked to some theoreticians, and they told me that it was a horrendous problem, and even if we did succeed in creating a model, there would be no way to tell if we were right."
A collaboration with a group from the National Institutes of Health also failed because the data from nuclear magnetic resonance experiments could not be interpreted for an RNA of the relatively large size that Draper had chosen to study.
Next, a postdoctoral student in his lab, Graeme Conn, offered to grow crystals of the RNA alone. That effort also failed, but then Conn decided to take the experiment an extra step by bringing the protein into play. At that point, crystals formed, and a group from the Biophysics Department, which included its chair, Professor Eaton Lattman, and his research associate Apostolos Gittis, was called upon to conduct the X-ray crystallography. The crystallographic technique finally allowed them to capture images and positions of atoms in the RNA molecule by unraveling X-ray diffraction patterns.
Draper's group worked in collaboration with Lattman's lab for nearly a year. At one point, they heard that another lab, at the University of Utah, was working on a similar project and was having success. Sensing the competition, Hopkins' scientists began to work night and day analyzing the data. In early April, the final image came into focus, and Draper prepared the announcement.
"People have been thinking that a lot of the important work in a cell depends on these kinds of tertiary folds in RNA," Draper said. "But until now only a few little bits of structure were known. What we have now is only 1 percent of the complete structure of the machinery of the cell that carries out protein synthesis--but it's a really interesting 1 percent. Looking at this machinery, this ribosome, we now have insight into what appears to be a general feature of cellular RNAs: Proteins have evolved to help the critical process of RNA folding."
With that knowledge, Draper said he can now return to previous hypotheses about the nature of RNA and see long-standing questions in a new light. The insight should advance his research quickly.
"It's like you've been working in a dimly lit room for a long time and somebody finally turns on a light," he observed. "Maybe it's just one corner of the room, but at last you can see what you've been stumbling over all these years. Now we just need to use that insight to extend our vision as far as we can."
This research was supported by the National Institutes of Health and the Wellcome Trust.