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For the first time, researchers are describing how the Rho protein actually stops gene expression.
New research has identified and described a cellular process that despite what the textbooks say has remained elusive to scientists until now – precisely how the copying of genetic material that, when started, is properly deactivated.
The discovery concerns a key process essential to life: the transcription phase of gene expression, which allows cells to live and do their jobs.
During transcription, an enzyme called RNA the polymerase wraps around the double helix of DNA, using one strand to match nucleotides to make a copy of genetic material – resulting in a newly synthesized strand of RNA that breaks when transcription is complete. This RNA enables the production of proteins, which are essential for all life and do most of the work inside cells.
As with any consistent message, RNA has to start and stop in the right place to make sense. A bacterial protein called Rho was discovered over 50 years ago due to its ability to stop or terminate transcription. In each textbook, Rho is used as a model terminator which, using its very strong driving force, binds to RNA and removes it from RNA polymerase. But a closer examination of these scientists showed that Rho wouldn’t be able to find the RNAs he needs to release using the manual mechanism.
“We started studying Rho and realized it couldn’t work the way people tell us it works,” said Irina Artsimovitch, study co-lead author and Ohio professor of microbiology. State University.
The research, published online by the journal Science today, November 26, 2020, determined that instead of attaching to a specific piece of RNA near the end of transcription and helping it relax from the DNA, Rho is “hitchhiking On RNA polymerase for the duration of transcription. Rho cooperates with other proteins to eventually bring the enzyme through a series of structural changes that end in an inactive state allowing the release of RNA.
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 throughout transcription.
“This is the first structure of a termination complex in any system, and was supposed to be unobtainable because it collapses too quickly,” Artsimovich said.
“This answers a fundamental question – transcription is fundamental in life, but if it was not controlled nothing would work. The RNA polymerase itself must be completely neutral. It must be able to make any RNA, including those that are damaged or that could damage the cell. By traveling with RNA polymerase, Rho can tell if the synthesized RNA is worth making – and if not, Rho releases it. “
Artsimovitch made many important discoveries about how RNA polymerase transcribes so well. She didn’t set out to counter years of understanding Rho’s role in termination until an undergraduate student in her lab identified surprising 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 have no RNA sequence 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, not even knowing if they are still attached to RNA polymerase.
In fact, scientific understanding of the Rho mechanism was established with the help of simplified biochemical experiments that often left out RNA polymerase – in essence, defining how a process ends without taking into account the process itself.
In this work, the researchers used cryo-electron microscopy to capture images of RNA polymerase operating on a DNA template from Escherichia coli, their model system. This high-resolution visualization, combined with high-end computation, made it possible to accurately model transcription termination.
“RNA polymerase moves around, corresponding to hundreds of thousands of nucleotides in bacteria. The complex is extremely stable because it needs to be – if the RNA is released it is lost, ”Artsimovitch said. “Still, Rho is able to bring down the complex in a matter of minutes, if not seconds. You can watch it, but you can’t get a stable complex to analyze. “
Using an intelligent method to trap complexes just before they collapsed allowed scientists to visualize seven complexes that represent sequential steps in the termination pathway, starting with Rho’s engagement with l ‘RNA polymerase and ending with a completely inactive RNA polymerase. The team created models based on what they saw and then made sure those models were correct using genetic and biochemical methods.
Although the study was conducted in bacteria, Artsimovitch said this termination process is likely to occur in other life forms.
“It seems to be common,” she says. “In general, cells use similar working mechanisms from a common ancestor. They all learned the same tricks as long as those tips were useful.
Reference: November 26, 2020, Science.
Artsimovitch, together with an international research team 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 Indian Government’s Department of Biotechnology; national institutes of health; and the Sigrid Jusélius Foundation.
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