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New research has identified and described a cellular process that, despite what the textbooks say, has remained elusive to scientists until now – precisely as the copy of genetic material that, once initiated, is properly deactivated.
The discovery concerns a key process essential to life: the transcription stage of gene expression, which allows cells to live and do their jobs.
During transcription, an enzyme called RNA polymerase wraps around the DNA double helix, using a strand to match nucleotides to make a copy of the genetic material – resulting in a newly synthesized strand of RNA that shuts down when transcription is complete. That RNA allows for the production of proteins, which are essential for all life and do most of the work inside cells.
Just like with any coherent message, RNA has to start and stop in the right place to make sense. A bacterial protein called Rho was discovered more than 50 years ago for its ability to interrupt or terminate transcription. In every textbook, Rho is used as a model terminator which, using its very strong motor force, binds to RNA and extracts it from RNA polymerase. But a closer look from these scientists showed that Rho would not be able to find the RNAs it needs to release using the textbook mechanism.
“We started studying Rho and realized it can’t work the way people tell us it does,” said Irina Artsimovitch, co-author of the study and a professor of microbiology at Ohio State University.
The research, published online by the journal Science today, November 26, 2020, determined that instead of latching onto a specific piece of RNA towards the end of the transcription and helping it unwind from the DNA, Rho actually “hitchhikes” the RNA. polymerase for the duration of transcription. Rho works with other proteins to eventually induce the enzyme through a series of structural changes that end in an inactive state that allows RNA to be released.
The team used sophisticated microscopes to reveal how Rho acts on a complete transcription complex made up of RNA polymerase and two accessory proteins that travel with it during transcription.
“This is the first structure of a termination complex in any system and it had to be impossible to achieve because it falls apart too quickly,” Artsimovitch said.
“It answers a fundamental question: transcription is fundamental to life, but if it were not controlled, nothing would work. RNA polymerase itself must be completely neutral. It must be able to produce any RNA, including those that are damaged or that could damage the cell. While traveling with the RNA polymerase, Rho can tell if the synthesized RNA is worth producing, and if not, Rho releases it. “
Artsimovitch made many important discoveries about how RNA polymerase successfully completes transcription. He didn’t decide to counter years of understanding about Rho’s role in the layoff until a college student in his lab identified striking mutations in Rho while working on a genetics project.
Rho is known to silence the expression of virulence genes in bacteria, essentially keeping them dormant until they are needed to cause infection. But these genes don’t have RNA sequences that Rho is known to preferentially bind to. For this reason, Artsimovitch said, it never made sense for Rho to only look for specific RNA sequences, without even knowing if they are still attached to the RNA polymerase.
Indeed, scientific understanding of the Rho mechanism was established using simplified biochemical experiments that often excluded RNA polymerase, essentially defining how a process ends without taking the process itself into account.
In this work, the researchers used cryo-electron microscopy to acquire images of the RNA polymerase operating on a DNA template in Escherichia coli, their model system. This high-resolution visualization, combined with high-end computation, made accurate modeling of transcript termination possible.
“RNA polymerase moves, coupling hundreds of thousands of nucleotides in bacteria. The complex is extremely stable because it has to be: if the RNA is released, it is lost, “Artsimovitch said.” Yet Rho is able to bring down the complex in minutes, if not seconds. You can look at it, but you can’t get it. a stable complex to be analyzed “.
Using a clever method to trap the complexes just before they fell apart allowed scientists to visualize seven complexes that represent sequential steps in the termination path, starting with Rho’s engagement with RNA polymerase and ending with a Completely inactive RNA polymerase. The team created models based on what they saw and then made sure these models were correct using genetic and biochemical methods.
Although the study was conducted on bacteria, Artsimovitch said this disruption process could occur in other forms of life.
“It appears to be common,” he said. “Generally, cells use similar working mechanisms from a common ancestor. They all learned the same tricks as long as these tricks were useful. “
Artsimovitch, working with an international research group of collaborators, co-led the study with Markus Wahl, a former Ohio State graduate student now at Freie Universität Berlin.
This work was supported by grants from the German Research Foundation; the German Federal Ministry of Education and Research; the Indian Council of Medical Research; the Department of Biotechnology, Government of India; the National Institutes of Health; and the Sigrid Jusélius Foundation.
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