For the first time, researchers described how the Rho protein actually inhibits gene expression.
New research has identified and described a cellular process that has remained elusive to scientists, despite what textbooks say – exactly how the copying of genetic material, once started, is properly closed. has gone.
The discovery concerns an important process for life: the transcription phase of gene expression, which enables cells to live and do their work.
During transcription, an enzyme is called Royal army Polymerase wraps itself around the double helix DNA, Using a strand to match nucleotides to make a copy of the genetic material – resulting in a newly synthesized strand of RNA that breaks down upon completion of transcription. That RNA enables the production of proteins, which are essential for all life and do most of the work inside cells.
Just as with any coherent message, RNA needs to start at the right place and stop to be understood. A bacterial protein called Rho was discovered for more than 50 years because of its ability to inhibit, or transcribe, transcription. In each textbook, Rho is used as a model terminator that, using its very strong motor force, binds RNA and ejects it from RNA polymerase. But a closer glance by these scientists revealed that the RHO would not be able to find RNA using the textbook mechanism.
“We started studying RHO and realized that it couldn’t possibly work in the way people work,” said Irina Artsimovich, co-lead author of Microbiology of Studies at Ohio State University.
The research, published online by the journal Science Today, November 26, 2020, determined that instead of attaching a specific piece of RNA near the end of transcription and helping to release DNA, the RHO is actually “irritant” on the RNA polymerase for the duration of transcription . RHO eventually cooperates with other proteins through a series of structural changes of proteins that end with the inactive state enabling the inactive state of RNA.
The team used sophisticated microscopes to reveal how Rho acts on a complete transcription complex composed of RNA polymerase and two adjuvant proteins that travel with it in transcription.
“This is the first structure of a termination complex in any system, and was considered impossible to obtain because it dissociates very quickly,” Artisimovich said.
“It answers a fundamental question – transcription is fundamental to life, but if it is not controlled, nothing will work. RNA polymerase by itself has to be completely neutral. It should be able to make any RNA, including those that are damaged or can damage the cell. When traveling with RNA polymerase, the RHO can tell if the synthesized RNA is worth making – and if not, Rho. “
Artsimovitch has made several important discoveries about how RTA polymerase successfully completes transcription. She did not face years of understanding of RHO’s role in Termination, until a graduate student in her lab identified surprising changes at RHO while working on a genetics project.
Rho is known to silence the expression of viral genes in bacteria, essentially keeping them inactive until they are required to cause infection. But these genes have no RNA sequence known to bind Rh preferentially. Because of that, Artsimovich said, it has never been understood that RHO looks only for specific RNA sequences without knowing if they are still associated with RNA polymerase.
In fact, a scientific understanding of the RH mechanism was established using simplified biochemical experiments that often bypassed RNA polymerase – in essence, defining how the process ends without factoring in the process.
In this work, the researchers used cryo-electron microscopy to capture images of RNA polymerase operated on a DNA template in their model system Escherichia coli. This high-resolution visualization combined with high-end computations enabled precise modeling of transcription termination.
“RNA polymerase moves together, combining thousands upon thousands of nucleotides in bacteria. The complex is very stable because it has to be – if RNA is released, it is lost, ”said Artisimovich. “Yet Rho complicates in a few minutes, if not seconds. You can see it, but you cannot find a stable complex to analyze. “
Just before being able to visualize seven complexes representing sequential steps in the termination pathway, starting from the engagement of RHO with Rh polymerase and ending with fully inactivated RNA polymerase, a cleft to trap complex. Using the method. The team created models based on what they saw and then ensured that these models were correct using genetic and biochemical methods.
Although the study was conducted in bacteria, Artsimovich stated that this termination process is likely to occur in other forms of life.
“It seems normal,” he said. “In general, cells use the same function mechanism from a common ancestor. They all learned the same trick as long as these tricks were useful. “
Reference: 26 November 2020, Science.
Artemimovich, working with an international research team of collaborators, is now co-leading the study with Ohio State alumnus Marcus Wahl at Frevy Universite Berlin.
This work was supported by a grant from the German Research Foundation; German Federal Ministry of Education and Research; Indian Council of Medical Research; Department of Biotechnology, Government of India; National Institute of Health; And the Sigrid Jusius Foundation.