Single Molecule Imaging Reveals Surprising Activity
Sua Myong, a member of the Precision Proteomics theme at the IGB, is in the business of shifting paradigms, one molecule at a time. Myong, who is also a member of the bioengineering faculty, uses imaging techniques such as FRET (fluorescence resonance energy transfer) to help understand what is happening to single molecules during various cellular processes.
Looking at cellular processes at the single molecule level enables researchers to see very detailed behavior that would be lost if one were looking at large numbers of molecules at a time. In several cases her work has resulted in a new understanding of a given molecule, including a project involving Rep helicase protein.
"We wanted to visualize Rep movement from the 3' end toward the 5' end (of the single-stranded DNA)," says Myong. "We already knew that Rep cannot unwind DNA as a monomer, so we expected Rep to dissociate from the ssDNA when it encountered the double strand DNA region."
But to her surprise, Myong's imaging techniques demonstrated that when the Rep helicase arrived at the junction where the single strand DNA (already unwound) meets the double strand DNA (dsDNA), the Rep molecule did not dissociate from the DNA but appeared instead to snap back to the 3' end and take the same journey... over and over and over again. In a series of elegant experiments using FRET, Myong demonstrated that, not only does the Rep helicase shuttle back and forth along the ssDNA, but it also is mediated by the 3' end of the ssDNA, somehow looping back toward the junction, which enables the helicase to zip back to the 3' end almost instantaneously.
Myong also determined how the shuttling movement took place. She demonstrated that ATP hydrolysis provided the fuel for the movement: when there was a high concentration of ATP, the shuttling movement was faster than when there was a low concentration.
Myong's work suggests that Rep protein has an auxiliary role, beyond unwinding DNA, though it is not completely clear what that role might be inside of a cell. One theory, says Myong, is that by shuttling along the ssDNA, Rep is preventing other, random proteins from attaching to the fairly "sticky" ssDNA. Myong did find that in the presence of Rep, the formation of RecA filament, which forms along the ssDNA during replication, is impeded.
These findings represent a paradigm shift in the understanding of Rep helicase and what its role is.
"It turns out that a single unit of Rep is a robust, repetitive translocase on ssDNA," says Myong of her findings. "It's likely that more units are needed to carry out the unwinding reaction."
In addition, a similar type of repetitive shuttling activity also has been found in at least five other helicases in different contexts, suggesting a paradigm shift in our understanding of helicases in general, says Myong.
More recently, Myong has contributed to figuring out how the RIG I protein recognizes a viral intruder. Researchers have known that RIG I is the first alert system for viruses and that the system works because RIG I somehow recognizes either the triphosphate tag that viruses have, or the double-stranded RNA, which also is characteristic of viruses. However, researchers did not know how RIG I worked mechanistically. Nor did they understand the role of the ATPase domain within RIG I. ATP is a little fuel cell; why did RIG I need fuel?
Myong used a technique called "protein-induced fluorescent enhancement," in which a fluorescent dye attached to a specific region of a molecule, glows with more or less intensity depending on how close it is to the protein that is interacting with that molecule. With that technique she and her colleagues discovered that RIG I moves—an activity that was not known before—and that it moves along the double-stranded RNA. In addition, the activity of RIG I along the RNA is greatly stimulated when the triphosphate tag on the 5' end of the intruding virus is present.
RIG I translocation explains the presence of ATPase domain, and the increase in activity in the presence of both dsRNA and the triphosphate tag suggests that RIG I relies on both pathogenic indicators to determine the presence of a virus, rather than just one or the other, says Myong.
"RIG I is double checking to really make sure it has both pathogenic signals, and that there really is a virus present," she says.
Myong fell in love with bench work at the University of California, Berkeley, where she earned both her bachelor's degree and her doctorate.
"I liked the bench work," she says. "Running gels, cloning. I always worked in a lab as an undergraduate." Myong's molecular and cellular biology bench work skills come in handy when she has to modify the protein she studies. For these experiments, a protein's extraneous binding sites are "silenced" without affecting the function of the protein and then desired binding sites are engineered, again without impacting the protein's function.
At the IGB, Myong helped build a single molecule fluorescence technique and fluorescence cell studies for the Precision Proteomics theme. The goal for the cell technique is to be able to image whole cells that have been tagged with fluorescence in a critical region and then, with enough spatial resolution, image the cells at the single protein level.
All of Myong's work up until now has been in vitro but she hopes to begin observing molecular processes in vivo. With these new tools and approaches, Myong stands to continue making surprising discoveries that will advance our understanding of a wide variety of cellular processes.