For the first time, researchers describe how rho protein really stops gene expression.
New research has identified and described a cellular process that, despite what textbooks say, has remained elusive to scientists – just as it properly turns off the copying of genetic material that once started.
The finding concerns a vital key process: the transcription phase of gene expression, which enables cells to live and do their jobs.
An enzyme is called during transcription RNA Polymerase wraps around the double helix of DNAUsing a strand to align nucleotides to create a copy of the genetic material – resulting in a newly synthesized strand of RNA that breaks off after transcription is complete. This RNA enables the production of proteins that are essential for all life and do most of the work in cells.
As 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 because it can stop or finish transcription. In every textbook, Rho is used as a model terminator, which binds to the RNA with its very strong motor force and pulls it out of the RNA polymerase. However, a closer look by these scientists showed that Rho would not be able to find the RNAs he needs to release using the textbook mechanism.
“We started studying Rho and found that it couldn’t possibly work the way people tell us,” said Irina Artsimovitch, co-lead author of the study and professor of microbiology at Ohio State University.
The research, published online by the journal science Today, November 26, 2020, it was discovered that, instead of attaching to a specific piece of RNA towards the end of transcription and helping it to unwind from DNA, Rho was actually hitchhiking to RNA for the duration of the transcription Polymerase. Rho works with other proteins to eventually coax the enzyme through a series of structural changes that end in an inactive state that allows the RNA to be released.
The team used sophisticated microscopes to show how Rho acts on a complete transcriptional 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 a system and should be impossible to maintain because it is falling apart too quickly,” said Artsimovitch.
“It answers a fundamental question – transcription is fundamental to life, but if it wasn’t 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 harm the cell. When traveling with RNA polymerase, Rho can determine whether the synthesized RNA is worth making – and if not, Rho releases it. ”
Artsimovitch made many important discoveries about how RNA polymerase completes transcription so successfully. She didn’t set out to counter years of understanding about Rho’s role in the resignation until a 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 inactive until needed for infection. However, these genes do not have RNA sequences that are known to preferentially bind Rho. Because of this, Artsimovitch said, it never made sense for Rho to just look for specific RNA sequences without knowing if they were still bound to RNA polymerase.
In fact, the scientific understanding of the Rho mechanism has been established through simplified biochemical experiments that often omitted the RNA polymerase – essentially to define how a process ends without considering the process itself.
In this work, the researchers used cryo-electron microscopy to capture images of RNA polymerase operating on a DNA template in Escherichia coli, their model system. This high-resolution visualization in combination with high-end calculations made it possible to precisely model the transcription termination.
“The RNA polymerase moves and matches 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, ”said Artsimovitch. “However, Rho is able to have the complex fall apart in minutes, if not seconds. You can look at it, but you cannot analyze a stable complex. ”
Using a clever method of capturing complexes just before they fall apart, the scientists were able to visualize seven complexes representing successive steps in the termination path, from Rho’s commitment to RNA polymerase to a completely inactive RNA polymerase. The team built models based on what they saw and then used genetic and biochemical methods to ensure that those models were correct.
Although the study was done on bacteria, Artsimovitch said this termination process will likely occur in other life forms.
“It seems common,” she said. “In general, cells share similar working mechanisms from a common ancestor. You’ve all learned the same tricks as long as those tricks were useful. ”
Reference: November 26, 2020, science.
Artsimovitch led the study in collaboration with an international research team of collaborators together with Markus Wahl, a former doctoral student from Ohio State who is now studying at Freie Universität Berlin.
This work was supported by grants from the Deutsche Forschungsgemeinschaft; the Federal Ministry of Education and Research; the Indian Council for Medical Research; the Department of Biotechnology, Government of India; the National Institutes of Health; and the Sigrid Jusélius Foundation.
