Study reveals secret of key cellular process disproves biology textbooks

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, once 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 polymerase wraps around DNA’s double helix, using one strand to match nucleotides to make a copy of genetic material – resulting in a newly synthesized strand of RNA that binds together 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 closer examination of these scientists showed that Rho would not 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 itself to a specific piece of RNA near the end of transcription and helping it relax DNA, Rho actually does “auto-stop” 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 to 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 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 startling 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 has to be – if RNA is released, it is lost,” Artsimovitch said. “Still, Rho is able to bring down the complex in minutes, even seconds. You can watch it, but you can’t get a stable complex to analyze.”

Using a smart method to trap the 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.”