In a landmark experiment at the Department of Energy’s SLAC National Accelerator Laboratory, scientists used a powerful X-ray laser to capture the first snapshots of a chemical interaction between two biomolecules in real time and at the atomic scale. This chemical interaction flips an RNA “switch” that regulates the production of proteins – the workhorse molecules of life.
The study demonstrates the groundbreaking potential of X-ray free-electron lasers (XFELs) for studying RNA, which guides the production of proteins in the cell, acts as the key genetic material in HIV and other similar retroviruses, and also plays an important role in most types of cancer. The results of the study have been reported in Nature.
This specific type of RNA switch called riboswitch can only be found in bacteria. As a result, a better understanding of its function may provide a new way to turn off the production of proteins and destroy harmful germs without causing any side effect in the humans they infect.
Previous experiments at SLAC’s X-ray laser have studied biological reactions like photosynthesis that are triggered by light. But this is the first to observe one that is triggered by the chemical interaction of two biomolecules in real time and at the atomic scale. This really demonstrates the unique capability that X-ray free-electron lasers offer that no current technology, or any other technology on the horizon, can do. It’s like you have a camera with a very fast shutter speed, so you can catch every move of the biomolecules in action.
Yun-Xing Wang, Structural Biologist, National Cancer Institute’s Center for Cancer Research
The experiments were performed at SLAC’s Linac Coherent Light Source (LCLS), a DOE Office of Science User Facility. They were the first to show how XFELs can capture real-time snapshots of a chemical flipping a biological switch and potentially make movies of biomolecules, including RNA, as they interact chemically. This finding provides a better insight into the fundamental workings of the cell that otherwise cannot be obtained.
Seeing RNA Shape Shifting
RNA, a major part of the genetic material in all living cells, comes in many different types that work together to guide protein manufacturing by the cell’s ribosomes, in accordance with the blueprints encoded in DNA. However, extensive regions present in both RNA and DNA do not code for any protein – the genetic “dark matter.”
For many years, researchers believed that these regions did not play any major role. Now, it is known that these regions actually play a key role in determining where and when the genes turn on and off and fine-tune their function. Wang informed that most forms of cancers are caused by mutations in these non-coding regions and hence a deeper insight into the workings of these regions is essential for both fundamental biology and cancer research.
However, it is not easy to figure out the activity of the RNA non-coding regions. As RNA molecules are flexible and wobbly, it is difficult to use them in large crystals that are often required to study their atomic structure at X-ray light sources. This barrier was overcome by LCLS, enabling scientists to obtain structural data from smaller nanosized crystals that can be made more easily.
LCLS’ powerful X-ray laser pulses are so short that they can collect data from each nanosized crystal in a few millionths of a billionth of a second, before the X-rays can cause damage. These X-ray laser pulses are billion times brighter than any available before.
Vibrio vulnificus is a bacterium that causes cholera. Wang’s team studied a riboswitch from this bacterium and observed that this riboswitch sits in a long strand of messenger RNA (mRNA) that copies the DNA’s instructions for protein production, so that these instructions can be read and carried out by the cell’s ribosome. The switch functions like a thermostat that regulates the production of proteins.
In this case, the mRNA guides the manufacture of a protein, which, in turn, helps to create adenine, a small molecule. When excess adenine is present in the bacterial cell, adenine molecules penetrate the riboswitch pockets, flipping the riboswitches into a different shape. This changes the speed at which adenine and proteins are produced.
First Stills of an Elegant Film
For the LCLS experiments, the scientists developed nanocrystals, which integrated millions of copies of the riboswitch, and then combined them with a solution that contained adenine molecules. The crystals were so small that adenine can uniformly and rapidly enter into every corner of them, penetrate riboswitch pockets, and flip them almost immediately, as if they were millions of synchronized swimmers performing a flawless move.
Once the mixing started, the team took snapshots of this chemical interaction by bombarding the crystals with powerful X-ray laser pulses at accurately timed intervals. This provided the first glimpse of a transitory intermediate stage in the process, which took place in just 10 seconds. The team also captured the first images of the riboswitch in its initial, empty-pocket state, and observed that it existed in two slightly different configurations, only one of which takes part in switching.
The scientists observed that the rapid change in the shape of the riboswitches was so remarkable that it also altered the shape of the entire crystal. Generally, such a significant change would damage the crystal and affect the experiment. However, these crystals were so tiny that they held on together, allowing the X-ray laser to obtain structural data from them.
To me it’s still a mystery how the crystal managed to do that. This actually opens up a lot of new possibilities and gives us a new way to look at how RNA and proteins interact with small molecules, so this is very exciting.
Soichi Wakatsuki, Professor, SLAC
Apart from SLAC’s LCLS and the National Cancer Institute, researchers contributing to this study came from Johns Hopkins University, Arizona State University, University of Hamburg, the Center for Free-Electron Laser Science at Deutsches Elektronen-Synchrotron (DESY), Hauptmann-Woodward Medical Research Institute, the DOE’s Argonne National Laboratory, and the National Institutes of Health.
The NIH Intramural Research Programs and the National Science Foundation funded the study.
SLAC National Accelerator Laboratory/Youtube.com