Cryo-EM reveals structural dynamics of RNA polymerase-DNA interactions during transcription initiation

Every living cell transcribes DNA into RNA. This process begins when an enzyme called RNA polymerase (RNAP) attaches to the DNA. Within a few hundred milliseconds, the DNA double helix unwinds to form a knot called a transcription bubble, so that an exposed strand of DNA can be copied into a complementary strand of RNA.

It’s not entirely clear how RNAP achieves this feat. A snapshot of RNAP opening this bubble would provide a wealth of information, but the process happens too quickly for current technology to easily capture visualizations of these structures. Now, a new study Nature Structural and Molecular Biology describes E. coli RNA polymerase opening the transcription bubble.

The results, obtained in 500 milliseconds of mixing RNA polymerase and DNA, shed light on the fundamental 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,” said the study’s first author, Ruth Saecker, a research scientist in Seth Darst’s lab at Rockefeller. “Understanding this process is crucial because it is a major regulatory step in gene expression.”

A unique view

Darst was the first to describe the structure of bacterial RNA polymerase, and studying its intricacies has remained a major focus of his lab. While decades of work have established that binding RNA polymerase to a specific sequence of DNA triggers a series of steps that open the bubble, how RNA polymerase separates the strands and positions a strand in its active site remains a hotly debated topic.

Early work in this area suggested that opening the bubbles slowed the process down considerably, dictating how quickly RNA polymerase could move on to RNA synthesis. Later results in this area challenged this idea, and many theories emerged about the nature of this rate-limiting step. “We knew from other biological techniques that when RNA polymerase first encounters DNA, it creates a set of highly regulated intermediate complexes,” says co-author Andreas Mueller, a postdoctoral researcher in the lab. “But this part of the process can happen in less than a second, and we weren’t able to capture structures on such a short time scale.”

To better understand these intermediate complexes, the team collaborated with colleagues at the Center for Structural Biology in New York, who developed a robotic inkjet system that can rapidly prepare biological samples for analysis by cryo-electron microscopy. Through this partnership, the team captured complexes forming within the first 100 to 500 milliseconds of the RNA polymerase-DNA encounter, producing images of four distinct intermediate complexes detailed enough for analysis.

For the first time, a clear picture of the structural changes and intermediates that form during the initial stages of RNA polymerase binding to DNA has emerged.

Technology was extremely important to this experiment. Without the ability to quickly mix DNA and RNA polymerase and capture an image of it in real time, these results would not exist.

Ruth Saecker, first author

Get into position

By examining these images, the team was able to describe a sequence of events showing how RNA polymerase interacts with the DNA strands as they separate, in a level of detail never before seen. As the DNA unwinds, RNA polymerase gradually latches onto one of the DNA strands to prevent the double helix from coming back together. Each new interaction causes RNA polymerase to change shape, allowing new protein-DNA connections to form. This includes the expulsion of part of a protein that blocks the DNA from entering the RNA polymerase active site. This forms a stable transcription bubble.

The team suggests that the rate-limiting step in transcription may be the positioning of the template DNA strand into the active site of the RNAP enzyme. This step involves overcoming significant energy barriers and rearranging several components. Future research will aim to confirm this new hypothesis and explore other steps in transcription.

“We only studied the very early stages in this study,” Mueller says. “We hope to then look at other complexes, later stages, and other steps in the transcription cycle.”

Beyond resolving conflicting theories about how DNA strands are captured, these results underscore the value of the new method, which can capture molecular events occurring in milliseconds in real time. This technology will enable many more such studies, helping scientists visualize dynamic interactions in biological systems.

“If we want to understand one of the most fundamental processes in life, which all cells perform, we need to understand how its progression and speed are regulated,” Darst says. “Once we know that, we’ll have a much clearer idea of ​​how transcription begins.”