Researchers at the U.S. Department of Energy’s SLAC National Accelerator Laboratory have discovered new insights into the basic mechanisms of RNA polymerase II (Pol II), the protein responsible for copying DNA into RNA. Their research shows how a protein adds nucleotides to a growing RNA strand. The results were published in: Proceedings of the National Academy of Scienceshas potential applications in drug development.

Pol II is found in all life forms, from viruses to humans. Its role in gene expression, the process by which genetic information is used to synthesize proteins, makes it one of the most important proteins in the cell. Understanding the exact mechanism by which RNA polymerase adds nucleotides to RNA has been a long-standing challenge for the scientific community. Previous studies have provided only partial insights into this process at low resolution.

One of the main difficulties in studying Pol II has been the interstitial nature of metals, particularly magnesium, in its active site. These metals play an important role in the chemical reactions that lead to the addition of nucleotides, but their short-lived presence makes observation difficult.

“The polymerase chemistry involves metals that are transient in the active site, making them difficult to see,” said co-author Guillermo Calero, a researcher and professor at the University of Pittsburgh. “This has become a significant obstacle to fully understanding the nucleotide addition process.”

To overcome these issues, the team used a new crystallization technique that involves a special salt known to promote interactions between proteins. This technique allowed the researchers to capture the polymerase in a state never seen before. This breakthrough allowed them to observe the “trigger loop,” the moving part of Pol II that positions nucleotides in the active site, in unprecedented detail.

Another key component of the study was the use of SLAC’s Linac Coherent Light Source (LCLS) X-ray laser, which allowed the researchers to collect data before the sample was exposed to severe radiation damage, providing a clearer picture of polymerase structure and function.

“For the first time, we were able to see three magnesium ions in the active region,” said co-author and SLAC scientist Aina Cohen. “This was only possible thanks to the free-electron laser data, which allowed us to see the third metal ion, which is extremely sensitive to radiation.”

Another interesting discovery came from studying a mutated version of Pol II. This mutant RNA polymerase works faster than the wild type, but also produces more errors.

“The mutation changes the structure of Pol II,” said study co-author Craig Kaplan, a professor at the University of Pittsburgh. “Using LCLS, we can identify these structural changes, which can reveal how the mutation affects Pol II activity.”

The team is working on time-resolved experiments to capture the real-time dynamics of the polymerase trigger cycle as it interacts with nucleotides, in the hope of unraveling the complexity of RNA polymerase function and contributing to a broader understanding of gene expression.

Additionally, by understanding the detailed mechanisms of human Pol II, researchers can now explore the design of molecules that can inhibit viral and bacterial polymerases while reducing harmful interactions with human polymerases. This is especially true in the field of drug discovery, where the goal is to develop drugs that are effective against pathogens but safe for human cells.

“These structures not only improve our understanding of how human RNA polymerase works, they also provide the basis for the development of more selective antiviral drugs with fewer side effects,” Cohen said.