When Richard Ebright scans through a biology textbook, many of the illustrations are intimately familiar, especially when he gets to the sections on transcription—the process by which DNA is rendered into RNA messages. "We can always tell what parts of pictures in these sections come from our work," he says.
Since his days as a graduate student in the laboratory of Jon Beckwith at Harvard University, Ebright has been fascinated with the cell's transcription machinery and how it kick-starts gene expression. Experiments he conducted at Harvard from 1981 to 1987 and at the Institute Pasteur in Paris, from 1985 to 1987 contributed to understanding how transcription factors recognize their binding sites on DNA. This process had been a black box until then.
After joining the faculty at Rutgers University in 1987, Ebright started asking more detailed questions about these proteins. "Some researchers focus on one experimental method and move from question to question. In contrast, we have stayed with one series of questions and applied different, increasingly more sophisticated, experimental methods to answer them," he explains.
One of the first questions on the list was, what does a transcription factor do when it gets to its DNA site? He used a variety of molecular genetics, biophysical, and structural biology methods to explore this issue, and by the mid to late 1990s had revealed basic principles of how a transcription factor that activates transcription sends its signal to the general transcriptional machinery. Ebright then turned his attention to the next black box.
During the late 1990s, Ebright shifted his primary focus to RNA polymerase, the component of the general transcription machinery responsible for synthesizing RNA. In particular, he wanted to understand how RNA polymerase starts the process of transcription (initiation), strings together nucleotides (elongation), and eventually comes off the DNA (termination). Three major advances that occurred in the late 1990s aided the study of these processes. The first was the determination of high-resolution structures of RNA polymerase, which allowed researchers to "see" how the enzyme is organized. The second was the development of methods to detect and characterize single molecules in solution, which allows researchers to "watch" individual molecules of the enzyme interact, move, and change conformation as they carry out reactions. And the third was the development of powerful methods to attach small probes, such as cross-linking groups and fluorescent groups, at specific sites within large biomolecules.
Using a method called fluorescence resonance energy transfer (FRET), Ebright's group placed pairs of fluorescent probes at strategic sites on RNA polymerase and then measured distances between the probes at different stages of transcription. "It's like figuring out how a car works. If you put a probe on a piston and a probe on the engine block, you could see how the piston moves relative to the engine block," he explains. "If you then repeat this process for each other key pair of engine components and drive train components, you could deduce the full operation of the engine and drive train."
In a paper published in Science in 2006, Ebright used FRET to show that transcription initiation by RNA polymerase involves DNA "scrunching"—a mechanism in which RNA polymerase remains stationary on the DNA and unwinds and pulls downstream DNA into itself and past its active center. In a separate paper published in the same issue of Science, Ebright confirmed these results using an independent method called single-molecule DNA nanomanipulation, which involved mechanically stretching and twisting a single DNA molecule attached at one end to a magnetic bead and at the other to a glass surface and detecting changes in twisting of the DNA as individual molecules of RNA polymerase bound to the DNA and performed transcription. Ebright is continuing studies of transcription initiation and now also is conducting studies of transcription elongation and transcription termination. He expects that a complete mechanistic picture of transcription will require 10 to 15 years to emerge.
Starting in 2000–2001, Ebright added a new research focus: small molecules that inhibit bacterial RNA polymerase. Such compounds provide valuable tools to analyze the structure and function of RNA polymerase. Many such compounds also have antibacterial activity, and some offer promise for developing broad-spectrum antibacterial agents and antituberculosis agents. The identification of new inhibitors, characterization of new inhibitors, and structure-based design and synthesis of novel analogs having higher potencies and improved pharmacological properties are increasingly important components of Ebright's research program. "This was not something that we planned when we started basic research on transcription and transcriptional regulation in the 1980s, but, remarkably, it turns out that the research now has clear practical applications," states Ebright, "and we are actively pursuing those practical applications."
When he is not in the lab, Ebright enjoys walking and running outdoors, finding his way with map and compass. He started orienteering—a sport in which participants use map and compass to navigate from point to point in diverse and unfamiliar terrain—only relatively recently with his two teenage children. But it has become a passion. If his dedication to his other passion, understanding transcription, is any indication, Ebright will continue pursuing challenging terrain for years to come.
