Biochemistry, Structural Biology
Cold Spring Harbor Laboratory
Dr. Joshua-Tor is also a professor at Cold Spring Harbor Laboratory.
Structure and Function of Nucleic Acid Regulatory Complexes
For a couple of days in 2004, Leemor Joshua-Tor and one of her graduate students knew something no one else did. They relished the moment, sitting side-by-side at the computer working out the details of their newly revealed protein structure. "'This is unbelievable,' we said to ourselves. 'We are the only two people on the planet who know this! We discovered Slicer.'" Recounting the story, Joshua-Tor's sentences are still punctuated by laughter and obvious pleasure.
By discovering the identity of the enigmatic Slicer, Joshua-Tor and grad student Ji-Joon Song solved a mystery in a newly discovered area of gene regulation: RNA interference (RNAi). Since then, Joshua-Tor, a structural biologist at Cold Spring Harbor Laboratory, has applied the same careful structural analysis to solve other important puzzles in gene regulation and even virus replication.
Although RNAi has existed for millions of years, and is now a popular tool for exploring biology, its existence was only revealed in the 1990s. Scientists discovered that small pieces of RNA, just 21–24 nucleotides long, had the power to silence gene expression. These small interfering RNAs (siRNAs) could pair with messenger RNAs (mRNAs) if their sequences matched. Once that pairing happened, a mystery enzyme, which researchers referred to as Slicer, cut the mRNA in two. With the mRNA destroyed, the cell could no longer copy it into protein, and the gene was effectively silenced.
Several research groups were hunting for the identity of the elusive Slicer protein with genetic and molecular biology techniques, but Joshua-Tor took a different tack. The field was converging on a protein called Argonaute as the likely candidate for Slicer. Joshua-Tor and Song used x-ray crystallography, which involved sending high-energy x-ray beams through protein crystals of Argonaute to generate a molecular image of the protein and determine its functional shape.
Once the researchers saw the shape of Argonaute, they knew they'd found Slicer. The protein looked like other enzymes that clip RNA in the cell. Moreover, their computer modeling showed a groove in the protein that could fit the paired-up siRNA and mRNA.
To prove that Argonaute not only looked like but actually was Slicer, Joshua-Tor collaborated with HHMI investigator Gregory Hannon to perform two critical experiments. The researchers made small mutations in the protein, right where the mRNA should be cut, and showed that the Slicer activity was lost. Then they expressed the human Argonaute protein in E. coli, a bacteria that normally lacks RNAi activity. Even then the human protein could cut mRNAs when siRNAs were present, showing that Argonaute was the only protein required for Slicer activity.
That experiment "put the nail in the coffin for people who were still doubters," says Joshua-Tor. With that first Argonaute structure in hand, her team began determining the unique features and roles of other Argonaute proteins that are part of other RNAi-silencing pathways. They are also working to uncover the structural details of Argonaute and of siRNAs that silence whole regions of chromosomes, an ability Joshua-Tor and collaborators revealed recently. Those large complexes can contain several large proteins, but Joshua-Tor is undaunted. "We're getting better at studying bigger blobs—and we never work in a vacuum. We always move back and forth between structure and biology."
Within a year of the Slicer triumph, Joshua-Tor's team deciphered the structure of a protein called E1 that is found in papillomavirus, a small DNA tumor virus that causes cervical cancer. Scientists knew that E1 is a member of a family of proteins, called helicases, that unwind the DNA helix during chromosome replication, but it was not clear how such proteins accomplished the task. The researchers soon revealed how a complex of six E1 proteins pulls a single strand of DNA through a channel in the protein complex, a likely paradigm for other helicases. Although a vaccine against cervical cancer, Gardasil, is now available, it is not effective against every strain of the virus, and it does not work in women already infected with papillomavirus. To move closer to a new therapy that can block replication of the virus, Joshua-Tor is teaming up with HHMI investigator Taekjip Ha at the University of Illinois at Urbana-Champaign to develop a step-by-step, millisecond-timescale molecular movie of how this helicase complex works.
Finding the answer to two major questions in about as many years was really amazing, says Joshua-Tor, who is dean of the graduate program at Cold Spring Harbor's Watson School of Biological Sciences. The day that stands out in her mind, however, is the one on which she gathered about 10 of her collaborators together to unveil Slicer's identity. "That was one of my best days in science."