Scientists unlock secrets of how a key protein converts DNA into RNA

Researchers at the Department of Energy’s SLAC National Accelerator Laboratory have uncovered new insights into the fundamental mechanisms of RNA polymerase II (Pol II), the protein responsible for transcribing DNA into RNA. Their study shows how the protein adds nucleotides to the growing RNA chain. The findings, published in Proceedings of the National Academy of Scienceshave potential applications in drug development.

The Pol II protein is present in all forms of life, 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 precise 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 and low-resolution insights into this process.

One of the major challenges in studying Pol II has been the transient nature of the metals, particularly magnesium, present in its active site. These metals play a crucial role in the chemical reactions that lead to the addition of nucleotides, but their fleeting presence makes them difficult to observe.

“Polymerase chemistry involves metals that are transient in the active site, making them difficult to see,” said Guillermo Calero, a researcher and professor at the University of Pittsburgh. “This has been a major obstacle to fully understanding the nucleotide addition process.”

To overcome these challenges, the research team used a new crystallization technique that uses a special salt known to promote protein interactions. This technique allowed the researchers to capture the polymerase in a previously unseen state. This breakthrough allowed them to observe the “trigger loop,” a mobile part of Pol II that positions nucleotides in the active site, in unprecedented detail.

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

“For the first time, we were able to see all three magnesium ions in the active site,” said Aina Cohen, a collaborator and SLAC scientist. “This was only possible because of 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 version, but also produces more errors.

“The mutation changes the structure of Pol II,” said Craig Kaplan, a professor at the University of Pittsburgh. “With LCLS, we can identify these structural changes, which could reveal how the mutation impacts Pol II activity.”

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

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

“These structures not only advance our understanding of how human RNA polymerase works, but they also provide a basis for designing more selective antiviral drugs with fewer unwanted side effects,” Cohen said.