Biography of Jennifer A. Doudna BIOGRAPHY

n the central dogma of molecular biology, DNA is transcribed into RNA, which then is translated into protein. Although RNA may be Iconsidered simply an intermediary between these two important biological molecules, RNA is much more than just a recipe for making proteins. In the 1980s, researchers showed that certain RNA molecules function as enzymes, a role previously attributed solely to proteins. Jennifer A. Doudna, Ph.D., Professor of Molecular and Cell Biology and Chemistry at the University of Cali- fornia, Berkeley, has devoted her scientific career to revealing the secret life of RNA. Using structural biology and biochemistry, Doudna’s work decipher- ing the molecular structure of RNA enzymes (ribozymes) and other functional RNAs has shown how these seem- ingly simple molecules can carry out the complex functions of proteins. In two landmark studies, Doudna and Jennifer Doudna in the laboratory with postdoctoral associate Richard Spanggord. BIOCHEMISTRY colleagues solved the crystal structures of two large RNAs, the P4-P6 domain of the Tetrahymena thermophila group I systems. Her parents, both academics in have supportive female mentors.’’ intron ribozyme (1) and the hepatitis the humanities with avid interests in Doudna feels fortunate to have had a delta virus (HDV) ribozyme (2). By de- astronomy, geology, and evolution, en- strong female role model like Panas- termining their molecular structures, her couraged her interests. They provided enko to help guide her early in her work has advanced the understanding of Doudna with science books, museum career. RNA’s biological function. In her Inau- visits, and her first ‘‘hands-on’’ science gural Article published in this issue of experience—a summer studying worms RNA Exploration Begins PNAS (3), Doudna describes how a spe- and mushrooms in the laboratory of After earning her bachelor’s degree in cial piece of hepatitis C viral RNA, professor and family friend Don chemistry from Pomona in 1985, called the internal ribosome entry site Hemmes, at the University of Hawaii Doudna went on to pursue her biochem- (IRES), hijacks the host cell’s machin- (Hilo). After reading The Double Helix, istry doctorate with Jack Szostak at ery and induces it to churn out viral James Watson’s account of his and Harvard University (Cambridge, MA). proteins. Francis Crick’s discovery of the struc- During this time, Doudna’s fascination Among Doudna’s numerous awards ture of DNA, Doudna was hooked on with RNA began to blossom. ‘‘Up until and accomplishments are the Searle science and desired to delve deeper into the late 1980s, it was known that RNA Scholar Award (1996) and the National the mysteries of the life sciences. was involved in protein synthesis, but Academy of Sciences Award for Initia- To indulge her scientific curiosity, the discovery that RNA could have cat- tives in Research (1999). She is also an Doudna studied chemistry at Pomona alytic activity really revolutionized the investigator with the Howard Hughes College (Claremont, CA) where she met whole field,’’ recalls Doudna. This real- Medical Institute and an American several people who had a profound ization that RNA might have much Academy of Arts and Sciences Fellow. impact on her research career. These greater functional activities than its role In 2002, Doudna was elected to mem- included chemistry professors Fred as messenger RNA (mRNA) or as part bership in the National Academy of Grieman, whose passion for quantum of the ribosome provided the impetus Sciences for her contributions to the mechanics was ‘‘infectious,’’ and Corwin for Doudna’s subsequent research ca- field of biochemistry. Hansch, whose intensity and love for reer. With Szostak, Doudna made her research was ‘‘inspirational.’’ Doudna first mark on the RNA field. Doudna Erupting Scientific Interests began her first scientific research at and Szostak (1) reported the reengi- Pomona, working in the laboratory of Doudna grew up in Hawaii amidst the neering of an RNA self-splicing intron volcanoes, lush tropical forests, and re- Sharon Panasenko, her undergraduate into a ribozyme, capable of copying an mote beaches of Hilo. These natural advisor. Panasenko was not only a RNA template. ‘‘It was exciting for us wonders instilled in her an awe and ap- superb scientist, said Doudna, but also because it suggested that RNA could preciation of nature. Although her im- led by example, showing that a woman function as a polymerase,’’ Doudna says. mediate and extended family had no could be successful in what some per- scientists, Doudna first became inter- ceive as a male-dominated academic

ested in science in high school when she world. ‘‘It’s a challenging job, especially This is a Biography of a recently elected member of the took her first chemistry class and partic- for women,’’ says Doudna. ‘‘The further National Academy of Sciences to accompany the member’s ipated in a science seminar series high- along I get in my career, the more I see Inaugural Article on page 16990. lighting the chemistry of biological how important it is for young women to © 2004 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0408147101 PNAS ͉ December 7, 2004 ͉ vol. 101 ͉ no. 49 ͉ 16987–16989 Downloaded by guest on September 25, 2021 Graduating with her doctorate in experiments, Doudna, along with Liz Investigating Viral RNA 1989, Doudna remained in Szostak’s Doherty and Rob Batey, showed that Although Doudna has a strong interest laboratory as a postdoctoral fellow to this motif was the most critical interac- in the basic structure and function of continue her studies on self-replicating tion for allowing the RNA to form the RNA molecules, much of her current RNAs. ‘‘In the course of this work, I structure that it does (6). Around the work involves viral RNAs. Determining became curious about what the RNA same time, her colleague at Yale, how these RNAs function could lead to structure might be that would allow it to Thomas Steitz, reported the existence of strategies to combat a host of viral dis- have this kind of activity,’’ says Doudna. an abundance of the same motif in the eases. Doudna’s laboratory has deter- This curiosity eventually led Doudna ribosome (7). ‘‘Even in a very large mined how the small, self-cleaving RNA to the University of Colorado (Boulder) RNA, a motif that was observed in the found in the hepatitis delta virus (HDV) and the laboratory of Thomas Cech, P4-P6 domain turns out to be probably is able to function. As a human patho- who received the 1989 Nobel Prize in the most important motif, energetically, gen, HDV is only coinfectious in pa- chemistry for discovering the catalytic tients who already have a hepatitis B properties of RNA. As a research fellow for folding the RNA,’’ says Doudna. Her work on the P4-P6 domain began to virus infection; patients with HDV su- in Cech’s laboratory, Doudna began perinfection develop a more severe form crystallizing RNA molecules in hopes of illustrate the structural similarities between ribozymes and protein enzymes. of hepatitis. obtaining a molecular portrait of these Doudna, Adrian Ferre-D’Amare, and unique structures. According to Kaihong Zhou published the crystal Doudna, Cech is a deeply insightful sci- structure of this viral RNA in 1998 (2). entist who had assembled an outstand- “Can we get Based on this structure, they proposed ing laboratory research team. ‘‘The that the HDV ribozyme uses a cytidine highlight of this time period was the enough information so base in the RNA to shuttle protons dur- late-night discussions with members of ing the reaction, a mechanism remark- the lab and the occasional intense brain- that we can understand ably similar to protein ribonucleases. storming sessions with Tom,’’ Doudna ‘‘The surprise was that RNA might be says. the chemical basis for able to use a mechanism that is much Characterizing Ribozyme Structures RNA’s many biological more similar to proteins than had been appreciated before,’’ says Doudna. In In 1994, Doudna joined the faculty at Yale University (New Haven, CT) as an functions?” 2004, Doudna, Zhou, and Ailong Ke assistant professor. She rose rapidly published additional information on the through the ranks, becoming Henry functional characteristics of this viral Ford II Professor of Molecular Biophys- RNA (11). In a series of studies similar to the Another focus of Doudna’s laboratory ics and Biochemistry in 2000. Early in P4-P6 studies, Doudna examined a type her tenure at Yale, Doudna and Cech involves a segment of viral RNA called of nonenzymatic RNA found within an published the crystal structure of the the internal ribosome entry site (IRES). RNA–protein complex called the signal P4-P6 domain of the Tetrahymena ther- According to Doudna, IRES is a ‘‘pretty mophila group I intron ribozyme (4). recognition particle (SRP). SRP is a amazing structure that’s basically able to ‘‘That was the first time anyone had macromolecular machine that recognizes grab the ribosomes of infected cells and seen what a large structured RNA proteins leaving the ribosome and hijack them for making viral proteins.’’ looked like other than tRNA,’’ Doudna shunts them across the membrane of the Her team is studying this in the hepatitis says. This study directly revealed that cell or the endoplasmic reticulum. C virus (HCV), but the mechanism may RNA, rather than being a spaghetti-like Doudna became ‘‘interested in [SRP] be common to a number of viruses, in- molecule, could have a defined shape. a few years ago because, as an RNA- cluding poliovirus. ‘‘I think this is the ‘‘This P4-P6 domain structure showed us focused lab, we wondered why this RNA project in the lab that has the greatest how RNA is able to pack helices to- is so important for protein recognition potential to lead to something that gether to form a three-dimensional and why it had been so highly conserved might have an impact on human health,’’ shape,’’ she says, ‘‘which is much more in evolution.’’ Doudna says. In a series of experiments, reminiscent of what we see in proteins Doudna and postdoctoral fellow Doudna and Jeff Kieft determined that than anybody had previously been aware Robert Batey went on to characterize HCV IRES RNA actually has a three- of for RNA.’’ the crystal structure of the signal- dimensional structure responsible for its In the studies that followed, Doudna, recognizing domain of SRP (8). In activity (12, 13). In a collaboration with Joachim Frank Jamie Cate, and Raven Hanna found addition to the basic structure, Doudna, at the State University of New York in that a core of five magnesium ions clus- Batey, and Brian Rha conducted an in Albany, Doudna characterized HCV tered in one region of the P4-P6 do- vivo experiment using a strain of bacte- main, forming a nucleus around which IRES RNA interaction with the small ria in which portions of SRP sequence the rest of the structure could fold (5). subunit of the ribosome by using cryo- were manipulated. They found that only ‘‘We presented that as analogous to electron microscopy (EM) (14). The what happens in proteins, where protein the most conserved part of the structure cryo-EM structures revealed that a mu- structures typically fold around a hydro- was necessary to support growth (9,10). tant form of IRES was unable to induce phobic core,’’ she says. ‘‘Here the core ‘‘Analogous to what we had been able a conformational change in the 40S is chemically different, but the principle to do with the P4-P6 structure,’’ says ribosome that normally occurs when of folding is similar.’’ Doudna, ‘‘we were able to use the crys- it binds to wild-type IRES RNA. This Also in the P4-P6 crystal structure, tal structure of the core of the SRP to conformational change in the 40S sub- Doudna observed several examples of a direct biochemical studies to test what unit of the ribosome occurs in the re- particular motif—abundant adenosines the most important interactions were gion where the ribosome binds to the in unpaired regions of the RNA struc- energetically for holding that structure mRNA strand. ‘‘Based on the cryo-EM ture. By using various mutagenesis together.’’ structures, the IRES RNA might actu-

16988 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0408147101 Marino Downloaded by guest on September 25, 2021 ally be functioning like a C-clamp on show that the IRES RNA is responsible ‘‘RNA world,’’ where early forms of BIOGRAPHY the ribosome to physically clamp the for bringing two initiation factors, eIF3 life were based almost entirely on ribosome onto the viral mRNA in the and eIF2, together on the ribosome. RNA or an RNA-like molecule. ‘‘Obvi- correct place to initiate translation,’’ Mutant IRES RNAs become trapped at ously, until we build a time machine, says Doudna. intermediate stages along the pathway we can’t really go back and look at to active 80S ribosomes because one or that,’’ says Doudna, ‘‘but what we CalifoRNiA both of these factors do not associate might be able to do in the laboratory In 2002, Doudna made another cross- properly with the smaller (40S) ribo- now is to find out, ‘Is it even plausible country move to accept a faculty posi- somal subunit. that RNA could catalyze a variety of tion at the University of California, Although determining the structure different kinds of chemical reactions?’’’ Berkeley, as Professor of Biochemistry and function of these RNA molecules Doudna continues, ‘‘If the answer is and Molecular Biology. Doudna, her may someday provide therapeutic tar- yes, then that makes it more likely that husband, and their 2-year-old son, whom gets against a number of viruses, Doud- she calls ‘‘the biggest experiment I ever na’s interest in them is more basic. ‘‘For the ‘RNA World’ hypothesis might ac- did,’’ are closer to their extended fami- me, the bigger question is, ‘Can we get tually be true,’’ although she says it is lies and to a resource essential to both enough information so that we can un- a hypothesis that can never truly be of their research—the synchrotron derstand the chemical basis for RNA’s tested. Nevertheless, she says, ‘‘We at the Lawrence Berkeley National many biological functions?’’’ she says. hope that we can get information that Laboratory. ‘‘It will be exciting to make meaningful will help us figure out whether that At Berkeley, Doudna has continued comparisons between the chemistry of idea is likely to be true or not.’’ to study IRES RNA. A curious aspect ribozyme reactions and what happens in Doudna is excited about this possibil- of IRES RNA is that it requires only a protein enzymes that carry out similar ity and continues to probe the mysteries few of the initiation factors normally reactions.’’ of RNA in hopes that the results will used to begin translation. In her Inaugu- If RNA molecules show a breadth of provide a glimpse of early life on this ral Article (3), Doudna, Ji Hong, and structure and function similar to pro- planet: ‘‘I think the idea that RNA Chris Fraser extend previous findings to teins, the findings may have major might have played a critical role in that suggest a reason why mutant IRES implications for one of the most funda- process is very tantalizing.’’ RNA stalls at particular points in the mental scientific questions—the origin BIOCHEMISTRY assembly of active ribosomes. Using of life. Scientists have postulated that Melissa Marino, RNA-based affinity purification, they there might have been an ancient Freelance Science Writer

1. Doudna, J. A. & Szostak, J. W. (1989) Science 244, 6. Doherty, E. A., Batey, R. T., Masquida, B. & 11. Ke, A., Zhou, K., Ding, F., Care, J. H. D. 692–694. Doudna, J. A. (2001) Nat. Struct. Biol. 8, 339– & Doudna, J. A. (2004) Nature 429, 201– 2. Ferre-d’Amare, A. R., Zhou, K. & Doudna, J. A. 343. 205. (1998) Nature 395, 567–574. 7. Nissen, P., Ippolito, J. A., Ban, N., Moore, P. B. & 12. Kieft, J. S., Zhou, K., Jubin, R., Murray, M. G., 3. Ji, H., Fraser, C. S., Yu, Y., Leary, J. & Doudna, Steitz, T. A. (2001) Proc. Natl. Acad. Sci. USA 98, Johnson, Y. N. L. & Doudna, J. A. (1999). J. Mol. J. A. (2004) Proc. Natl. Acad. Sci. USA 101, 4899–4903. Biol. 292, 513–529. 16990–16995. 8. Batey, R. T., Rambo, R. P., Lucast, L., Rha, B. & 13. Kieft, J. S., Zhou, K., Jubin, R. & Doudna, J. A. 4. Cate, J. H., Gooding, A., Podell, E., Zhou, K., Doudna, J. A. (2000) Science 287, 1232–1239. (2001) RNA 7, 194–206. Golden, B., Kundrot, C., Cech, T. R. & Doudna, 9. Batey, R. T., Sagar, M. B. & Doudna, J. A. (2001) 14. Spahn, C. M. T., Kieft, J. S., Grassuci, R. A., J. A. (1996) Science 273, 1678–1685. J. Mol. Biol. 307, 229–246. Penczek, P., Zhou, K., Doudna, J. A. 5. Cate, J. H., Hanna, R. H. & Doudna, J. A. (1997) 10. Batey, R. T. & Doudna, J. A. (2002) Biochemistry & Frank, J. (2001) Science 291, 1959– Nat. Struct. Biol. 4, 553–558. 41, 11703–11710. 1962.

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