A group of scientists from the Scripps Research Institute for the first time in the world visualized the formation of cellular "protein factories" - ribosomes. In addition to the fact that the method developed by the group is in itself a great technical achievement, the work can open new directions in the development of antibiotics and the treatment of diseases associated with errors in the formation of ribosomes. In addition, these methods can be used to solve other complex problems in cell biology.
The discovery and observation of the molecules that form the ribosomes - the cellular factories that produce the proteins necessary for life - has been, for decades, the main, but seemingly inaccessible, goal of biologists. However, a new work of scientists from the Scripps Research Institute, published in the journal Science, made it possible to visualize the intermediate chemical stages of the process of their formation, or in the language of biochemists, their biogenesis.
"For me, it was an experiment of my dreams," says project leader James Skripps, a member of the Skaggs Institute for Chemical Biology and Dean of the Graduate School of the Scripps Institute, Professor James Williamson,. "Our wonderful colleagues from Scripps are ready to go to some crazy experiments, and when they work - it's just great".
Earlier studies of intermediate molecules (intermediates) that combine to form ribosomes and other cellular components have been severely limited by the possibilities of imaging techniques. Electron microscopy has long been possible to obtain photographs of molecules of this size, but this usually requires their preliminary cleaning. But in order to clear something, it is necessary to identify something first. In general, this means that scientists should have made preliminary conclusions about what kind of intermediates they were, than they could observe the actual process of their assembly.
"My laboratory was intensively working on studying the process of assembling ribosomes for about 15 years," says Williamson. "The basic steps were planned about 30 years ago. What no one really understood, it's how things happen in cells ".
A group of scientists from the National Institute for Scripps under the guidance of Professors Clinton Potter and Bridget Carragher, in collaboration with postgraduate students at the Kellogg School of Science and Technology, Anke School of Science and Technology Anke Mulder and Craig Yoshioka developed a new method, described in Science, called Discovery single-particle profiling (DSP), which removes the problem of purification and allows the successful visualization of untreated samples. The system of automatic data collection and data processing developed by scientists gave them the opportunity to decipher the resultant complex "mess" of data, which would otherwise be impossible.
For this project, a researcher at the Williamson Laboratory, Andrea Beck, isolated the ribosome components from the cells of Escherichia Coli, a bacterium commonly used for research. Then she chemically tore them apart, thus creating a solution of the small components that make up the ribosomes. The components were mixed, and then quickly colored and examined under an electron microscope. "We started with" dirty "samples that contained a terribly complex mixture of a wide variety of particles," Williamson commented.
Anke Mulder collected and analyzed the particles formed during the assembly reaction of the ribosome. To ultimately obtain the image of molecules, scientists processed more than one million electron microscope images of the assembly, using the computational algorithm that they created.
They received images from which it was possible to compose, as from pieces of a puzzle, parts of a ribosome. This was a convincing proof of the consistency of their methodology. "Regardless of how the data was processed, we always saw the same thing, and it made us believe that everything is happening just like this," says Williamson.
Additional confirmations appeared when researchers obtained images of ribosomal components corresponding to different periods of assembly reaction time.
After the destruction of the components, they prepared the samples at a different time distance from the start of the process, leaving enough time for the molecular mixture to begin assembling, as happens when assembling ribosomes in cells.
Having obtained a series of images corresponding to different assembly periods, scientists were able to show that as the reaction proceeds, the concentrations of larger and complex molecules increase, and smaller ones decrease. These results coincided with previously available, but limited information on the temporal nature of the ribosome formation process, once again confirming the success of the group.
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