Recent discoveries show how RNA polymerase interacts with DNA to initiate transcription, captured in milliseconds using advanced microscopy techniques. The breakthrough provides new insight into the mechanisms of gene expression regulation, helping to resolve a long-standing debate in the field.

Every living cell copies DNA into RNA. This process begins when the enzyme RNA polymerase (RNAP) binds to the DNA. Within a few hundred milliseconds, the DNA double helix unwinds, creating a transcription bubble that allows an open DNA strand to be copied into a complementary RNA strand.

How RNAP achieves this feat is largely unknown. A snapshot of RNAP as it opens up would provide a wealth of information, but the process is too fast for current technology to easily visualize these structures. Now a new study Nature Structural and Molecular Biology describes E. coli RNA during the opening of the transcription bubble.

The results, obtained within 500 milliseconds of RNAP being mixed with DNA, shed light on the basic mechanisms of transcription and answer long-standing questions about the initiation mechanism and the importance of its various steps. “This is the first time anyone has been able to capture transient transcription complexes as they form in real time,” says lead author Ruth Saeker, a research scientist in Seth Durst’s lab at Rockefeller. “Understanding this process is critical because it is a key regulatory step in gene expression.”

An unprecedented view

Durst was the first to describe the structure of bacterial RNAP, and uncovering its complexities remains a major goal of his lab. Decades of work have established that RNAP binding to a specific DNA sequence triggers a series of steps that pop the bubble, but exactly how RNAP separates the strands and positions one strand in its active site is still hotly debated.

Early work in this area showed that the opening of the bubbles acts as a critical slowdown, determining how quickly RNAP can proceed to RNA synthesis. Later results in the field have challenged this view, and various theories have emerged about the nature of this rate-limiting step. “We knew from other biological methods that RNAP forms a highly organized group of intermediate complexes when it first encounters DNA,” says co-author Andreas Müller, a postdoctoral researcher in the lab. “But this part of the process can happen in a fraction of a second, and we were not able to capture the structures in such a short time.”

To better understand these intermediate complexes, the team collaborated with colleagues at the Center for Structural Biology in New York, who have developed a robotic jet system that can rapidly prepare biological samples for cryo-electron microscopic analysis. Through this partnership, the team captured the complexes that form in the first 100 to 500 milliseconds of RNAP encountering DNA, providing images detailed enough to analyze four different intermediate complexes.

For the first time, a clear picture of the structural changes and intermediates that occur in the early stages of RNA polymerase binding to DNA has emerged. “This technology was extremely important for this experiment,” says Saeker. “Without the ability to rapidly mix DNA and RNAP and image them in real time, these results would not be available.”

Take a position

By examining these images, the team was able to outline the sequence of events that shows in unprecedented detail how RNAP interacts with DNA strands as they separate. As the DNA unwinds, RNAP gradually grabs one of the DNA strands to prevent the double helix from rejoining. Each new interaction causes the RNAP to change shape, allowing more protein-DNA bonds to form. This involves pushing out part of the protein that prevents DNA from entering the RNAP active site. This creates a stable transcription bubble.

The team proposes that the rate-limiting step in transcription may be the location of the DNA template strand in the active site of the RNAP enzyme. This step involves overcoming significant energy barriers and rearranging various components. Future studies will aim to confirm this new hypothesis and investigate other stages of transcription.

“We were only looking at the first steps of this research,” Müller says. “We then hope to look at other complexes, later time points, and additional steps in the transcription cycle.”

In addition to resolving conflicting theories about how DNA strands are captured, these results highlight the value of a new method that can capture molecular events occurring within milliseconds in real time. The technology will enable many more studies of this kind and help scientists visualize dynamic interactions in biological systems.

“If we want to understand one of the most fundamental processes in life—what all cells do—we need to understand how its progression and rate are regulated,” Durst says. “Once we know that, we will have a clearer picture of how transcription starts.”