A (Scientific) Lifetime Affair With Nucleic Acids.

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Published in final edited form as: J Mol Biol. 2025 Mar 12;437(11):169088. doi: 10.1016/j.jmb.2025.169088 A (Scientific) Lifetime Affair With Nucleic Acids Juli Feigon 1 Author information Article notes Copyright and License information 1 Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095-1569, United States ✉ Correspondence to : feigon@mbi.ucla.edu Issue date 2025 Jun 1. PMC Copyright notice PMCID: PMC12904028  NIHMSID: NIHMS2145405  PMID: 40086689 The publisher's version of this article is available at J Mol Biol Abstract I am Distinguished Professor in the Chemistry and Biochemistry Department at University of California, Los Angeles, where I was hired in 1985 as the first female assistant professor in the department. I received my PhD from University of California, San Diego, under the guidance of Professor David Kearns, where I used NMR spectroscopy to study drug binding to random sequence DNA and published the first two-dimensional NMR spectra of short synthetic DNA duplexes. From 1982 to 1985 I was a Damon Runyon-Walter Winchell Postdoctoral fellow in the Professor Alexander Rich laboratory, where I investigated structures of Z-DNA by NMR. At UCLA, my lab pioneered the application of macromolecular NMR spectroscopy to the study of DNA and RNA structure, folding, and interactions with cations, drugs, and proteins. We published the first NMR structures of DNA triplexes, quadruplexes, and aptamers, and our work has provided fundamental insights into DNA A-tract bending, cation interactions with DNA, Hoogsteen base pairs, and drug binding to DNA. My lab has made major contributions to understanding RNA folding, dynamics, and function, including pseudoknots, aptamers, ribozymes, and riboswitches, and recognition of RNA by proteins. Over the past 2 decades, the Feigon laboratory pioneered structure–function studies of telomerase, from solution NMR and X-ray crystal structures and dynamics studies of RNA and RNA–protein domains of human and Tetrahymena telomerase, to the first structure of a telomerase holoenzyme, by negative stain EM in 2013, and subsequent cryo-EM structures of telomerase and associated proteins. Recent work also includes structural biology of 7SK RNP. Keywords: NMR, cryo electron microscopy, G-quadruplex, telomerase, aptamer Biographical Overview I grew up in Highland Park, Illinois, a suburb of Chicago, in what was then a middle class neighborhood. My father was second generation American; his mother and father had emigrated from Tauragnai, Lithuania in 1911 and Belogorodka, Ukraine in 1907, respectively, when Jews were subject to pogroms and there were limited opportunities for jobs or even food. They met in the US, settled in Chicago and ran a grocery store and had two sons, Gershon and my father Edward (Isadore). My father applied to University of Chicago for college, but was rejected, in his view due to the Jewish quotas at the time. He started college at Illinois Institute of Technology but WWII intervened. After the war he went to UCLA on the GI bill, where he majored in chemistry and fell in love with California. After UCLA, my Dad returned to Chicago where he worked at a company called Py-O-My, which made baking mixes and pans. While there, he developed the first blueberry muffin mix ( Figure 1 ). My grandfather on my mother’s side immigrated to the US from South Africa where he was born; he was a chemist and started a successful company making paint. My maternal grandmother was second generation American with Latvian and Hungarian roots. My parents married young, my mother was 19 year’s old, and they had 3 daughters of which I am the middle. My mother Jackie was an indifferent housekeeper and a talented artist, and I remember going to art fairs with her. Unusually for that time, my parents divorced when I was four, and a year later my father re-married to my stepmother, Toby, whom he stayed happily married to for the rest of his life. We spent every other weekend with my Dad and Toby, my dad adopted her two kids, and they had a child together. A few years later my Dad was given the opportunity to move back to California as Vice President of new products at Dole Pineapple in San Jose. After that move, we spent school years with my mom and summers in California where combined we were 6 siblings! My stepmother loved us as her own, and I felt fortunate to experience two very different family lives, one more conventional with working dad and stay at home nurturing mom and the other a single working mom who expected us to be responsible for ourselves and have dinner on the table when she got home from work. Figure 1. Open in a new tab An advertisement for my father’s innovative bluebery muffin mix. I was always a good student and an avid reader (which also served as a way to avoid socializing). I was considered an ‘over achiever’, a categorization that my mother found ridiculous. As a child I loved running and gymnastics, and in junior high school I had the school record for the 500 yard ‘walk and run’, which involved running 3 ½ times around the tennis courts, and I competed in track meets where I generally won. But at that time high school did not have competitive sports teams for girls. There was no track and field and there was a gymnastic club but not team. I was fortunate to live in a school district with a top rated high school and good science classes. Besides science I was interested in creative writing, especially poetry and art, and in high school I was active in stage crew including set design. Most of the things I enjoyed as a child continue as hobbies today – gardening, now with a focus on native plants; cooking and farmers markets; reading, mostly novels; and exercising, including running and lifting weights – while my creative abilities especially in writing have been important contributors to my success as a scientist. Undergraduate school at Occidental College in Los Angeles (1972–1976) When it came time to apply to college, all but one of the schools I applied to was in California, including the small liberal arts college Occidental in Los Angeles, which offered me a full scholarship. So despite being admitted to Berkeley, I chose to go there. Since my father was a food chemist and my grandfather on my mother’s side was a paint chemist, it might seem natural that I majored in chemistry in college. However, I only recall thinking about this in retrospect, rather than as an influence on my choice of major at the time. Despite my interest in science, I was not sure, like my male science major friends were, that I wanted to be a scientist. I was also interested in writing and considered myself a poet. I was one of only two women in my graduating chemistry class of less than 30, and the entire chemistry department faculty was male. One advantage of being a female science student was that there were no expectations of me to do well (except from myself), and I didn’t really worry about a career. Although I was a straight A student in chemistry I was never encouraged by my chemistry professors to go on to an academic career in science; rather, since I was also a gifted writer and had received awards for my poetry, my professors suggested that I become a science writer. At that time, biochemistry was taught by the Biology Department, and I was fortunate to have a new faculty member Dr. Laura Mays (later Hoopes), teach the one quarter of biochemistry I had as an undergraduate. It was in that class (and a quarter I did research in her lab looking for changes in tRNA during aging) that I fell in love with nucleic acids, which have remained my research passion throughout my career. I remember very clearly how amazing I thought the structure of tRNA was, from the cloverleaf secondary structure to how it transformed to the L-shape tertiary structure seen in the crystal structure of tRNA (which had only been solved the year before). 1 , 2 I also clearly recall a lecture Dr. Mays gave on nucleosomes, where she explained the very new discovery that the DNA was on the outside of the histone core, which was just being figured out at that time based on neutron diffraction of deuterated nucleosomes. 3 (This was 2 decades before the crystal structure was solved. 4 ) Graduate school in chemistry and biochemistry at UCSD in La Jolla (1976–1982) I accepted UCSD’s offer for graduate school without ever visiting. After a summer running in the LA smog (and doing research in an Occidental college chemistry lab on Friedel-Crafts acylation reactions), I have a vivid recollection of arriving to the sun sparkling off the ocean in beautiful La Jolla. For my PhD research, I wasn’t sure what field I wanted to end up in but I was sure I wanted to study nucleic acids. I did rotations in three labs that worked on nucleic acids: David Kearns, a physical chemist doing NMR on tRNA, who became my PhD advisor ( Figure 2 ); John Abelson, a molecular biologist working on tRNA sequencing, who later helped me with letters of recommendation when I applied for postdoc fellowships; Bruno Zimm, a physical chemist studying DNA as a polymer; and Susan Taylor, a protein biochemist. In 1971, David, together with Dinshaw Patel and Robert Shulman, had published the first observation of imino proton resonances of tRNA in H 2 O by NMR at 220 MHz. 5 Figure 2. Open in a new tab David Kearns in his office at UCSD when I was a graduate student. Despite being supercompetitive I never heard David resent another scientists success. My first publication in the Kearns’ lab was an NMR study of DNA imino proton resonances in nucleosome core particles. 6 This project came out of my PhD candidacy (oral) exam, where I had proposed an original research project to determine whether the positively charged histone ‘tails’ bound in the grooves of the DNA. I learned that nucleosomes could be purified from chicken blood (avian red blood cells are nucleated) by nuclease digestion. So I and a friend went to a chicken farm to extract blood from chickens. David’s lab had a 300 MHz Varian correlation NMR spectrometer, which he had actually mortgaged his house to place the order for while waiting for his grant to be funded! I observed two broad imino proton peaks, one for A–T and one for G–C base pairs, and comparison to DNA extracted from the nucleosome core particles revealed almost no differences in the spectra. Thirty-nine years later much more elegant solid state NMR experiments by Chris Jaroniec and Hashim Al-Hashimi on nucleosome core particles with 13 C, 15 N-labeled DNA looking for A–T Hoogsteen base pairs also revealed only small spectral differences with the free DNA 7 ! Shortly after the nucleosome work, William (Bill) Denny, a medicinal chemist from Auckland, New Zealand, arrived in David’s lab for a sabbatical. The bulk of my thesis work was made possible by him ( Figure 3 ). First, he provided dozens of DNA-binding antitumor drugs whose interaction with my 146 base pair chicken DNA purified from nucleosome core particles I investigated by NMR, determining if they were intercalators or groove binders and how tightly they bound. 8 I spent many days sitting at the NMR spectrometer acquiring 1D correlation spectra. Second, Bill and a Swiss postdoc in the lab, Werner Leupin, started chemical synthesis of DNA. I was lucky to be given a couple of these DNA decamers to study by NMR. By this time the macromolecular NMR renaissance had started with the introduction of pulse and Fourier transform NMR, publication of the first COSY spectrum in 1976 9 and the first NOESY spectrum in 1980, 10 soon followed by developments in multidimensional NMR experiments to assign resonances and determine protein structures. 11 California Institute of Technology had a 500 MHz NMR spectrometer as a Regional NMR Facility, and I was able to get ~5 hours on it to collect data. I and John Wright, the department NMR facility manager, drove there from UCSD and collected a 2D NOESY spectrum. Our JACS communication (1982) was the first publication of two-dimensional NMR spectra of DNA 12 ( Figure 4 ). From the NOE crosspeaks and COSY spectra (acquired at UCSD on a 360 MHz NMR spectrometer with modified Varian console and Nicolet computer that allowed pure phase spectra) it was immediately clear that it should be possible to assign the spectra of short DNA duplexes, as subsequently shown. 13 – 15 I remember the excitement of presenting my work on a poster at the Xth ICMRBS (International Conference on Magnetic Resonance in Biological Systems) at Stanford in August 1982. Many of the people I met there became good friends as well as scientific competitors. Shortly after that conference I left for my postdoc position at MIT. Figure 3. Open in a new tab Juli as a young graduate student in the Kearns lab with visiting medicinal chemist William (Bill) Denny. Figure 4. Open in a new tab Stacked plot of 500 MHz NOESY spectrum of DNA decamer d(ATATCGATAT) duplex, the first published two-dimensional NMR spectrum of double-stranded DNA. 12 Postdoctoral fellow in Alex Rich’s lab at MIT (1982–1985): Z-DNA years At the time that I was in graduate school, if you wanted to get into a good lab for post doc it was recommended to start looking at least a year in advance since top labs generally expected you to apply for postdoctoral fellowships. I knew I wanted to continue to study nucleic acids, but I wasn’t sure if I wanted to stay in NMR. I also thought it was important to go somewhere outside of California. I loved California and thought that I wouldn’t be able to get a faculty job at a university where I had gone to graduate school or did my post doc training, so I didn’t want to eliminate another west coast school. Alexander Rich from MIT Biology Department had visited UCSD shortly after his paper on Z-DNA – the first crystal structure of a DNA duplex! – had appeared in 1979. 16 Later, I made an appointment to talk to Alex at a meeting we were both attending, where Alex immediately started telling me about what to write for my fellowship applications. So I looked up fellowships and wrote my applications and eventually received a Damon Runyun-Walter Winchell postdoctoral fellowship. Besides my interest in nucleic acids, I chose Alex’s lab since I wanted to learn some biology, I wanted to go to Boston (Cambridge) since it was considered the scientific center of the universe (or at least the US), and the Francis Bitter National Magnet Lab at MIT had a homebuilt 500 MHz NMR that I could use. After living in La Jolla for almost 6 years, Cambridge MA was quite a change. I moved into the upstairs apartment of a house in what was then not upscale Central Square with two other Rich lab members ( Figure 5 ), and every day walking to campus I passed homeless people, something I had never seen before. Scientifically Cambridge was an exciting place to be. In addition to all the famous scientists at Harvard and MIT, each summer many visitors would pass through the lab on their way to Gordon Conferences in New Hampshire. Alex’s lab was about 30 people, almost all postdocs, who virtually all came with fellowships. My fellowship proposal was to purify Z-DNA binding proteins and investigate their interactions with Z-DNA. I soon learned that there were no identified Z-DNA binding proteins in the Rich lab (it was another 10 years before any Z-DNA binding proteins were found. 17 ) But the lab did have a plentiful supply of short DNA oligonucleotides, synthesized by Gijs van der Marel in the laboratory of Jacques van Boom, a pioneer of DNA oligonucleotide synthesis by phosphotriester method, 18 in Leiden. Alex was pretty laissez-faire in how he ran the lab, so I had the freedom to do whatever science I wanted. Andy H.-J. Wang, a senior member of the lab (later he became a professor at U. Illinois and subsequently Vice President of Academia Sinica in Taiwan), took me under his wing and encouraged me to start investigating Z-DNA by NMR at the Magnet Lab. And Robert (Bob) Griffin, a leading solid state NMR spectroscopist, who at that time was a member of the Magnet Lab (later he became an MIT professor in the chemistry department) kept encouraging me to use the 500 MHz solution state NMR spectrometer. I decided I was more likely to eventually get an academic job if I investigated Z-DNA by NMR than trying to establish its biological roles. I was lucky to have easy access to the 500 MHz spectrometer at the Magnet Lab a 15 min walk away, which was the highest field available at that time, and I was equally lucky to gain the mentorship of Bob Griffin. The spectrometer was not only homebuilt with a pumped magnet but also had its own software, so whenever I wanted to try something new I had to cajole David Ruben to write the pulse program for me. I spent so much time at the Magnet Lab that I became an honorary member of the Griffin lab and eventually was given a desk space in his over-crowded lab. I published two papers on NMR of Z-DNA, the first in Nucleic Acids Research 19 and the second in Science (1985), 20 both of which are still being cited. Since I was the only person doing NMR in the Rich lab, I was almost completely on my own for the experiments, data analysis, and manuscript writing. I am most proud of the NAR paper on the B- to Z-DNA transition of d(m 5 CGm 5 -CGm 5 CG), where m5 is methyl at the 5 position of C, where I observed chemical exchange cross peaks between the two conformations in NOESY spectra run with a long mixing time of 1800 msec ( Figure 6 ). I used these exchange crosspeaks to assign the Z-DNA peaks based on my sequential assignments of the B-DNA form. This was one of the first observations by NMR of exchange between two conformations of a macromolecule in solution. Figure 5. Open in a new tab Thanksgiving Dinner with Rich lab friends Mike McMillan, Eileen Lafer, Ferran Azorin, visiting friend Keith Marzullo from my Occidental College days, me, and Kai Lowenhaupt. Did I mention I love to cook? Figure 6. Open in a new tab (A) One-dimensional slices from a NOESY spectrum of d(m 5 CGm 5 CGm 5 CG) under conditions where it is an approximately equimolar mixture of B-DNA and Z-DNA. Autocorrelation and exchange crosspeaks between the B-DNA and Z-DNA CH6 and GH8 resonances are labeled. The B-DNA assignments could thus be transferred to Z-DNA peaks. (B) The DNA duplex studied. (C) Handwritten letter from the new editor of Nucleic Acids Research (Sarah Elgin) accepting our paper with minor changes. The reviews came typed on carbon copies. 19 After two years in the Rich lab, it was time to start applying for faculty positions. At that time, a typical postdoc was only two or three years. I sent out about 40 applications and got 6 interviews. When I got the first interview offer, Mary Barkley, who was on sabbatical in the Griffin lab, generously arranged for me to give a practice talk. Some of my male colleagues told me I was only getting all those interviews because I was a woman, and said much the same thing when I got four offers. Much later, when I was elected to the National Academy of Sciences in 2009, similar comments were made (although not to my face). I mention this because of course there is an element of truth, so such comments can fester in insecure people like myself. On the other side of the coin, though, it can be equally true that this recognition would have happened sooner if I wasn’t a woman! Academic career at UCLA (1985–today) Getting started. I arrived at UCLA in November 1985 and moved into a small house that I had rented a few miles away from UCLA that came complete with a cat, and I found a housemate to help pay the rent. My lab was in a building called the Molecular Biology Institute (MBI; now Boyer Hall) that had come into being through the efforts of its first director and later Nobel laureate Paul Boyer, who saw the need to bring together scientists in this new field from across the campus. Being housed in the MBI was considered an honor (which included having to get tenure in the MBI to stay there, as well as in my department, Chemistry and Biochemistry). Richard Dickerson, who reported the first B-DNA crystal structure, had recently taken over as director and had been given 3 faculty positions to fill which included one for the then hot new field of macromolecular NMR spectroscopy. Since the MBI was in Life Sciences and Chemistry and Biochemistry in Physical Sciences, during my early years I had two deans. Funding for a 500 MHz NMR spectrometer, which by then was commercially available, came from the Life Sciences Dean while the rest of my startup package came from Chem and Biochem. While setting up my biochemistry lab and teaching for the first time I also needed to order my NMR spectrometer. Having no experience with choosing equipment and with spending so much money, this was extremely stressful for me. My spectrometer was delivered about a year later, and it took about another year to get everything working properly ( Figure 7 ). Figure 7. Open in a new tab My first NMR spectrometer from GE with Oxford magnet, then located in the basement of Young Hall. This ~40 year old magnet is still in use, but with a Bruker console. My appointment as an Assistant Professor was the first for a woman in the Chemistry and Biochemistry department. Luckily for me, Joan Valentine had been hired a couple of years earlier as a full professor in inorganic chemistry. She recognized I was struggling with insecurity about succeeding in my job and helped me a lot psychologically in my early years. Jane Strouse, who had gotten her PhD from Professor Frank Anet (a pioneering small molecule NMR spectroscopist in the organic division) and was director of the department NMR facility, was extremely helpful in setting up my NMR room and the NMR spectrometer installation (despite the fact that I bought a GE NMR instead of Bruker like all the departmental instruments, inadvertently assuring that I would not have to share my instrument with departmental users). Looking back to those times, although women faculty were rare, women staff , including teaching, administrative, and scientific, were not, and formed a largely unsung, dedicated, talented, and underpaid backbone to the department. Two years after I arrived, Sabeeha Merchant was hired as assistant professor (see her article in this special JMB Pioneers of Molecular Biology issue 21 ). I thought it was important to pay forward the support I had gotten from Joan, and did my best to help her when she arrived. In the event, Sabeeha was much more politically astute and able to take care of herself than I was, and ended up helping me! I had applied for an NIH R01 grant before I left MIT, and amazingly it was awarded, starting a few months after I arrived at UCLA. However, I was given funding for only 3 years instead of the usual five (to allow me to prove myself)! This meant I would need to get my first renewal before I was up for tenure (I succeeded). I was extremely fortunate that Andy Wang had given me samples of DNA oligonucleotides that had been synthesized in the Jacques van Boom lab, and I was able to include some preliminary results in the grant application and had experiments ready to go right away. Meanwhile, commercial DNA synthesizers using solid state phosphoramidate chemistry became available, and we were soon able to purchase one and make our own DNA oligonucleotides. I got my first graduate student (Dara Gilbert) and my first postdoc (Ponni Rajagopal, from Brian Reid’s lab) and about one or two new graduate student per year for the next few years. My little group was tight-knit and hardworking––although there were frequently more people in the lab at 10 at night than 10 in the morning—and I am grateful to all of them for their willingness to work for a woman ‘boss’, something that was not a given in those days ( Figure 8 ). I had little experience in being a mentor, and was happily surprised to discover how gratifying it was to have a group. By 1987, I finally had data for my first paper from my own lab 22 and presented it as a poster at the ‘Sarma meeting’ (Conversations in Biomolecular Stereodynamics) in Albany, New York. There I had the great good fortune to meet Vladimir (Vladi) Sklenar, a visiting postdoc in the Ad Bax lab from the then communist Czechoslovkia, who was working on NMR pulse sequence development for water suppression and on DNA studies. He became a frequent lab visitor and close collaborator for the next 15 years and a lifelong friend, as described by Ad Bax and myself in his recent scientific obituary. 23 Figure 8. Open in a new tab Party at Juli’s house ~1990 with my first group of graduate students Ken Addess, Dara Gilbert, Roman Macaya, Flint Smith, Edmond Wang, Robert Peterson (only his shoulder and ear is showing); postdocs Jung Hee Lee and Peter Schultze (back right); and Vladi Sklenar (sitting between Juli and Dara). Key Discoveries DNA–drug complexes and DNA triplexes Early work in the Feigon laboratory focused on NMR studies of non-B-DNA structures, specifically drug-DNA complexes, DNA triplexes, and B-Z junctions. The Rich laboratory had determined the crystal structure of echinomycin, a bis-intercalator cyclic hexadepsipeptide, in complex with DNA; surprisingly, the two central A-T base pairs bracketed by two aromatic rings from two bound echinomycin were in the Hoogsteen conformation. Solution NMR studies of the same complex by Dara Gilbert showed that the Hoogsteen base pairs were unstable, observed as increasing line broadening of imino proton resonances in NOESY spectra as a function of increasing temperature. We proposed that they were exchanging between Hoogsteen and an open or Watson-Crick paired state. 24 Thirty years later, my brilliant friend Hashim Al-Hashimi rigorously quantified Hoogsteen–Watson-Crick base pair exchange by R1rho relaxation in this system, 25 following his discovery in 2011 of a Watson Crick–Hoogsteen dynamic equilibrium in DNA duplexes in general. 26 Meanwhile, my first postdoc Ponni Rajagopal was studying DNA triplexes formed by mixing homopurine d(GAGAGAGA) and homopyrimidine d(TCTCTCTC) DNA strands. At that time, there was interest in such DNA triple helices formed by one homopurine and two homopyrimidine strands because of evidence that they formed in plasmids in a pH and superhelical density dependent fashion and therefore might occur in vivo 27 and also because of the potential use of triplexes in forming highly sequence-specific complexes for use in chromosome mapping 28 and in repressing transcription. Early fiber diffraction and UV studies had provided evidence for triple helices from poly(U) and poly (A) (or dU and dA) 29 and a base pairing scheme of Hoogsteen–Watson–Crick U–A–U triples had been proposed. A similar base triple for C + –G–C would require protonation of the Hoogsteen paired C imino, thus the pH dependence. Ponni was able to show formation of the predicted triplexes at low pH 5.5 by NMR and directly observe the protonated C + imino in the Hoogsteen base pairs, providing the first NMR evidence for DNA triplex formation. 30 Around that time, I realized that I would never get tenure if I didn’t start giving talks at meetings. In those days it was not usual for graduate students or postdocs to give talks at meetings, the exceptions usually being people sent there in their advisor’s place. (In fact, it wasn’t until I was an assistant professor that I could even get accepted to attend the then highly oversubscribed Nucleic Acids Gordon conference.) So I screwed up my courage and talked to one of the organizers (David Live) of the prestigious Keystone “Frontiers of NMR in Molecular Biology” conference that was set to take place in Park City, Utah in January 1989; I (out of desperation) told him that we had interesting results and he had no women speaking at his meeting. Thankfully, he did invite me, and my talk was amazingly well received. After my talk, people asked where we were submitting the DNA triplex work, and it gave me the confidence to send it to Science , where it got accepted. 30 I spoke in an evening session with the one other woman speaker at the meeting, and it changed my life. After that, I got frequent invitations to speak at meetings. In my early years doing macromolecular NMR, the field was not very friendly to women (it was also pretty harsh to people in general). I remember speaking at meetings in Europe where I was the only woman speaker and young women telling me it was the first time they had heard a women give a talk at a meeting. Thankfully, that has changed and the NMR community is now extremely supportive of young people and women and each other. The following year in 1989, Vladi Sklenar came to my lab for 3.5 months (NIH let me fund his postdoc salary and travel on my grant). 23 I had two projects ready for him, but we only got to the first. We worked together on a DNA oligonucleotide that had been designed to fold into an intramolecular triplex. Since it had potential C + –G–C as well as T–A–T base triples, triplex formation was expected to be pH dependent. We put the sample in an NMR tube and collected a series of imino proton spectra as a function of pH, and I vividly remember how we immediately could see spectral changes that indicated a transition from an intramolecular triplex to intramolecular duplex with single strand overhang, with the two conformations in slow exchange as we raised the pH ( Figure 9 ). We ended up with a short Nature paper (called a letter in those days) 31 and a JACS communication 32 from that 3.5 month visit. I should mention that a Nature paper in those days was generally two pages long ( Nature ‘letter’) and was considered to be a preliminary report of important results, so follow up work with more details was usually published in other journals like Biochemistry or JMB . Figure 9. Open in a new tab One-dimensional imino proton spectra of dGAGAGAACCCCTTCTCTCTTTCTCTCTT (HD28), where italics indicate loop nucleotides in the folded triplex, as a function of pH. At pH 5.5 imino proton resonances for 7 Watson-Crick and 6 Hoogsteen base pairs are visible between 12–16 ppm, as well as low-field shifted protonated C aminos. As the pH is raised, new resonances appear corresponding to a partial duplex with single strand. At pH 7.0 there is about 50:50 mixture of the two conformations shown at right. The T–A–T and C + –G–C triples are shown at upper right. 31 Studies of DNA triplexes continued to be a major focus of my lab in the early years. We published many more papers on other intramolecular DNA triplexes, that contained different sequences and loops, non-natural bases (in collaboration with Peter Dervan), 33 , 34 or an RNA third strand, 35 including one in Science by graduate student Roman Macaya with Dara Gilbert and undergraduate Shiva Malek on the effect of mismatches in the third strand. 36 With two Science and one Nature paper in 3 years plus other papers I was able to get tenure. Later, in collaboration with Stephan Grzesiek, we applied his newly developed method to detect scalar couplings due to hydrogen bonds between imino proton donors and acceptor nitrogens in Watson–Crick base pairs in RNA 37 to directly detect N–H•••N hydrogen bonds in both Watson–Crick and Hoogsteen base pairs in DNA triplexes. 38 Subsequently we applied similar methods to directly detect hydrogen bonds in G-quartets. 39 DNA quadruplexes and aptamers In the 1990s we got interested in the structures formed by G-rich telomere repeat sequences. The propensity for GMP to form G-quartets had been proposed 25 years earlier and a model for a four-stranded G-quadruplex proposed based on fiber diffraction of poly(G). 40 , 41 In the late 1980′s, Sen and Gilbert reported that G-rich regions of immunoglobulin switch regions formed parallel four-stranded structures on polyacrylamide gels 42 and Jamie Williamson as a postdoc in Tom Cech’s lab biochemically characterized G-quadruplex formation in various telomere repeat oligonucleotides. 43 We decided to work on telomere repeats d(TTTTGGGG) from Oxytricha nova (now Sterkiella nova ), using 1.5 repeats (Oxy1.5) and 3.5 repeats (Oxy-3.5). The first 4 Ts were deleted to avoid having 5′ ‘tails’. Graduate student Flint Smith struggled with assignments by 1 H NMR; the NOESY spectra indicated that some guanines were syn , and sequential assignments only worked for B-DNA. So Flint substituted inosine one by one at each of the guanines. I remember telling Flint that he would know his assignments were correct when all the NOEs made sense. There was one NOE that could not be explained unless the T4 loops went diagonally across the end G-quartets. Our 1992 paper on the dimeric and unimolecular G-quadruplexes formed by Oxytricha telomere repeats d(G 4 T 4 G 4 ) (Oxy-1.5) and d(G 4 TG 4 T 4 G 4 T 4 G 4 ) (Oxy-3.5) including a model of the ‘diagonally looped’ Oxy-1.5 44 ( Figure 10 ) was published as a short paper in the same issue of Nature as an article reporting the crystal structure of d(G 4 T 4 G 4 ) from Alex Rich’s lab. 45 The differences were discussed in a highlight. 46 (Fortunately I was not aware of this competition ahead of time). In contrast to our Oxy-1.5 model with diagonal loops, the crystal structure, that was solved by molecular replacement with a model, had the expected edge loops. I had just gotten tenure, and I was worried about the effect of these conflicting results on my hard earned good reputation. We proceeded to publish follow up papers that showed unambiguously that our structure was correct, 47 leaving open the possibility that the different structures might be due to the different cations (Na + vs K + ) used. I was really happy when Voet and Voet put our G-quadruplex structure in their Biochemistry textbooks in the first new edition after it was published. Figure 10. Open in a new tab NMR structures of (A) dimeric Oxy-1.5 G-quadruplex (sequence dG 4 T 4 G 4 ) with two diagonal loops, looking into medium width groove (PDB 156D), (B) unimolecular Oxy-3.5 G-quadruplex (sequence dG 4 T 4 G 4 T 4 G 4 T 4 G 4 ) with one diagonal and two edge loops (PDB 230D), and (C) thrombin binding aptamer (sequence dGGTTGGTGTGGTTGG) with two G-quartets and three short edge loops (PDB 148D). (D-F) Schematic of Oxy-1.5 G-quadruplex (D), Oxy-3.5 G-quadruplex (E), and thrombin binding aptamer (F). Syn guanines are gray and anti guanines are white. Loops for Oxy-1.5 and Oxy-3.5 are TTTT. (G) Schematic of G-quartets found in Oxy-1.5 and Oxy-3.5. Numbers correspond to top G-quartet in Oxy-1.5. (H) Schematic of G-quartets found in thrombin binding aptamer. Numbers correspond to top G-quartet. Groove widths, syn and anti glycosidic torsion angles, and relative strand orientation vary with the structures and are determined by the loop fold (diagonal vs edge). Oxy-1.5 and Oxy-3.5 are mixed parallel and antiparallel strands while the thrombin binding aptamer is all antiparallel. 44 , 55 , 62 , 154 A decade later, Martin Horvath and Stephen Neidle published crystal structures of Oxy-1.5 in Na + 48 and K + , 50 respectively, that were the same as our solution structure in Na + . 44 By then we had also shown that this sequence adopts the same fold regardless of monovalent cation. 49 Meanwhile, many other G-quadruplex structures formed from telomere repeat and other sequences containing runs of Gs had been solved that formed parallel, antiparallel, or mixed inter- or intramolecular quadruplexes with various loops including ones that crossed a groove from one end to the other as well as other unexpected arrangements. 51 Another exciting quadruplex structure that we solved by NMR was the thrombin-binding DNA aptamer that binds to and inhibits human α-thrombin 52 ( Figure 10 ). In vitro selection or SELEX (selective evolution of ligands by exponential enrichment) of RNAs (or DNAs) for specific target binding or function had been developed independently in 1990 by the Szostak and Gold labs, and the name aptamer was coined for the RNA (or DNA) product of the selection process. 53 , 54 The thrombin-binding aptamer, dGGTTGGTGTGGTTGG, studied concurrently by graduate student Roman Macaya in my lab and by Phil Bolton’s lab, was the first DNA aptamer structure to be determined. 55 – 57 It forms two G-quartets with the loops in a chair-like formation along the edges ( Figure 10 ). [A crystal structure of the thrombin-binding aptamer in complex with thrombin had traced the backbone incorrectly. 58 , 59 ] More than 30 years later, our first thrombin aptamer paper is still being cited, including 22 times in the past 12 months. Many other aptamers have since been found to contain G-quadruplexes and/or been developed as sensors or therapeutics. Today, quadruplexes have been proposed to act as inhibitors of telomerase activity, play a role in mechanism of telomere repeat synthesis, and to be unwound or prevented from forming by single-stranded telomeric DNA binding proteins. An RNA quadruplex has also been proposed to form at the 5′ end of telomerase RNA and in the telomere RNA TERRA. 60 Beyond telomeres and telomerase, numerous roles have been proposed for DNA and RNA G-quadruplexes in cancer, disease, and gene regulation, 61 everything from amyloid to aptamers to SARS CoV2 RNA, and they are also of interest in nanotechnology. I am proud that we correctly determined a previously unknown fold for the first ‘high-resolution’ structure of a G-quadruplex, at a time when NMR methods for accurate determination of nucleic acids structures were still being established, 44 , 62 and for the first discovery of the presence of G-quadruplex structures in some aptamers. 55 Cation binding to nucleic acids Due to the nature of nucleic acids, which contain a phosphodiester backbone with a negative charge for each nucleotide, cations play critical roles in their structures and stabilities. We realized this early on with our studies of DNA triplexes, which in general require divalent cations to stably form due to the close approach of phosphates between the Hoogsteen-paired pyrimidine strand in the major groove and the purine strand. Formation of G-quadruplexes requires monovalent cations like Na + or K + , which bind within or between quartets, respectively, associating with the unpaired electrons at the guanine carbonyls, and K + greatly increases G-quadruplexes stability vs Na + . 40 Postdoctoral fellow Nick Hud spearheaded a thermodynamic analysis by NMR that not only determined how many cations bound but also elegantly showed that the preferred coordination of K + over Na + in G-quadruplexes is actually driven by the greater energetic cost of Na + dehydration with respect to K + dehydration, rather than a more optimal fit to coordination sites formed by G-quartets. 63 Nick also recognized that by using 15 N-labeled ammonium ion N 15 H 4 + , which has the same van der Waals radius as K + , the number of cation binding sites in G-quadruplexes could be directly observed and binding sites located, and together with Vladi Sklenar developed experiments that could monitor their movement through the G-quadruplex and out into solution 64 , 65 ( Figure 11 ). Figure 11. Open in a new tab Monitoring NH 4 + (ammonium ions) in a DNA quadruplex. (A) N Z -exchange HSQC spectra of Oxy-1.5 with NH 4 + at two different mixing times, showing 1 H and 15 N chemical shifts corresponding to the inner (I), outer (O) and bulk (B) NH 4 + autocorrelation and cross-peaks (autocorrelation peak for bulk NH 4 + at 19.8 ppm not shown). These spectra were used to determine the lifetime of the inner ion (red in structure below) (B) Portion of a 1 H ROESY spectrum showing the diagonal and cross peaks of three NH 4 + resonances, corresponding to the inner (red), outer (gold), and bulk (blue) NH 4 + resonances, with arrows indicating their movement from one site to the next. The structure of the quadruplex with corresponding NH 4 + is shown at right. 64 , 65 N 15 H 4 + also proved useful in studying monovalent cation binding to the minor groove of DNA duplexes in general and DNA A-tracts in particular, 66 although here binding is in fast exchange (where the peaks shift corresponding to their relative populations) rather than the slow exchange observed in G-quadruplexes (where two separate sets of peaks appear). A-tracts are runs of 4 or more A–T base pairs without at TpA step. 67 Multiple A-tracts repeated in phase with the helical turn results in macroscopically observed bending as manifested by anomalous migration on polyacrylamide gels. 68 The origin of bending in DNA containing A-tracts was hotly debated. 69 At that time, crystal structures of B-DNA, specifically the ‘Dickerson dodecamer’ d (CGCGAATTCGCG), suggested there were waters in the minor groove that formed a two-layer ‘spine of hydration’. 70 , 71 Furthermore, it was widely believed that monovalent cations bound to DNA in a delocalized manner without sequence specificity. 72 Our NMR studies with various DNA duplexes showed that N 15 H 4 + localized to the narrow minor groove of A-tract sequences (which progressively narrows in the 5′ → 3′ direction). 66 These results were supported by higher resolution crystal structures of DNA, which showed a layer of Na + beneath the waters in the minor groove (cations and water could not be distinguished at the lower resolution of the earlier studies). 73 Our work showed that sequence-specific binding of cations in the minor groove of A-tracts could contribute to sequence-directed curvature of DNA. Later, visiting graduate student Richard Stefl from Vladi’s lab expanded on this work to provide the structural basis for why a TpA step disrupts A-tract bending while ApT does not, showed that multiple repeats of A-tracts cause bending out of the plane to form a left-handed superhelix, and showed a monovalent cation type dependence of bending. 74 We had finally solved a major conundrum of DNA A-tract bending! For RNA, both monovalent and divalent cations play critical roles in folding and stability. For various RNAs, we probed for divalent cation binding site by NMR using Mn 2+ , Mg 2+ , and Co NH 3 6 3 + and monovalent sites using NH 4 + . 75 , 76 One interesting example of Ca + binding to a riboswitch is briefly discussed below. The RNA world: RNA aptamers and riboswitches In the early 1990s, I started to get interested in studying RNAs. I realized that biologically relevant RNA could adopt many more diverse structures than DNA. At that time, just determining the structures of RNA tetraloops by NMR was a significant accomplishment and of major interest to RNA scientists. 77 , 78 We started with proton NMR studies of an HIV-1 Rev binding element RNA that had been identified by in vitro selection in Jack Szostak’s lab. 79 Dave Bartel, who was then in Jack Szostak’s lab, trained my graduate student Robert Peterson to do in vitro transcription of RNA. In 1992 the Williamson and Pardi labs published methods to make 13 C, 15 N-labeled oligonucleotides by using 13 C, 15 N-labeled NTPs purified from E. coli , which could then be used to enzymatically synthesize labeled RNA using T7 RNA polymerase and synthetic DNA templates as developed by Olke Uhlenbeck in the late 1980s. 80 – 82 In collaboration with Vladi Sklenar, whose annual visits to my lab were always times of great productivity and fun, over the next decade starting in 1993 we developed a series of NMR pulse sequences and methods for assigning 13 C, 15 N-labeled RNA and DNA oligonucleotides, 83 – 86 and started applying them to determine structures of aptamers, 87 , 88 GAAA tetraloop receptor, 89 hairpin ribozyme loop B, 90 and other biologically interesting RNAs. Vladi always worked with lab members including Robert Peterson and postdocs Thorsten Dieckman and Sam Butcher, who made samples and sat by him at the spectrometer while he showed them how to get the signals he was looking for. Robert Peterson later became my long time NMR facility manager and implemented many pulse sequences for the lab. 91 Sam Butcher had received his PhD in an RNA biochemistry lab studying the hairpin ribozyme, and had come to my lab to learn NMR of RNA while we benefited from his expertise of the RNA world. In 1996, the first three-dimensional RNA aptamer structures were reported, for binding ATP, FMN, arginine and citrulline, all determined by NMR. 92 The structure of the ATP aptamer was determined concurrently by Thorsten Dieckmann in my lab 88 and by Dinshaw Patel’s lab. 93 The binding target ATP had been used as part of the Szostak lab plan to create aptamers that could do chemistry. It was really exciting to see how the ATP aptamer folded to form an RNA-binding pocket for ATP ( Figure 12 ). Many new principles of RNA folding appeared from these RNA aptamer studies. Figure 12. Open in a new tab NMR structure of the ATP aptamer with bound AMP. Structure, sequence with conserved nucleotides circled, and schematic of the fold and interactions are shown. Unusual G–G (G7–G11, G17–G30) base pairs (yellow) stack on each stem (green) to buttress the binding pocket, and the AMP is recognized by three conserved nucleotides (magenta). Gray rectangles indicate stacking interactions. 88 At that time, methods were still being developed to accurately calculate nucleic acids structures from NMR data, including correct interproton distances and optimal numbers of restraints to go from a ‘model’ to a ‘structure’, and each lab had their own protocols. My postdoc Peter Schultze had realized that during structure calculations with the distance geometry program we were using (DISGEO) 94 the chirality of the sugars, e.g. at the 2′ position where ribose has a hydroxyl group, could flip, so he had written a program to restrain them to correct chirality. Not realizing this turned out to be a problem for many of the published nucleic acids structures, since a change in chirality at the 2′ position changed ribose into arabinose, including many of the riboses in the ATP aptamer from Patel lab, as we discovered when we compared the structures from the two labs. We subsequently explained the presence of chirality errors in many nucleic acids structures in the PDB in a letter to the editor published in Nature , 95 I (nervously) explained it at a Keystone Frontiers of NMR meeting, Peter’s program was incorporated into DISGEO, and the PDB started checking for correct stereochemical nomenclature at deposition. Our ATP aptamer structure was later featured in Lehninger Principles of Biochemistry as an example for in vitro selection. Riboswitches, which contain naturally occurring aptamer domains, were discovered a couple of years later. 96 , 97 Riboswitches are widely distributed in bacteria and are generally found in the 5′-UTR of mRNAs where they bind specific metabolites to regulate transcription or translation. In 2009, postdoctoral fellow Mijeong Kang determined the first NMR solution structure of a riboswitch aptamer, from the preQ1 riboswitch (after synthesizing the preQ!). 98 PreQ1 is a biosynthetic precursor of queuosine, a hypermodified guanine nucleotide found in the anticodon of some tRNAs. The 36 nt preQ1 aptamer forms a hairpin followed by a single-stranded A-rich tail in the absence of preQ1, but in the presence of preQ1 folds up to form an unusual H-type pseudoknot enclosing the preQ1. Concurrently, a crystal structure of the preQ1 bound aptamer domain was reported. 99 This provided us a unique chance to compare the solution NMR and crystal structures of the ligand-bound aptamer. Working with Mijeong, postdoctoral fellow Qi Zhang found small but significant conformational differences in the positions of residues above the binding pocket, suggesting conformational heterogeneity in the ligand-bound state. 100 Qi’s detailed NMR study included rigorous dynamics studies that showed that the observed conformational differences between the solution and crystal structures of the B. subtilis preQ 1 riboswitch aptamer were largely due to the presence of Ca 2+ in the crystallization conditions, in particular one Ca 2+ in a binding pocket that forms on ligand binding but that our studies showed is not important for folding or ligand recognition. This work not only provided unique information on ligand binding and aptamer conformational dynamics, but also set the standard for accurate and ‘high resolution’ solution NMR studies of RNA. Subsequently we determined the structure and investigated conformational dynamics of the even more challenging 59 nt (20 kD) class II preQ1 riboswitch aptamer, 101 which again was determined to comparable accuracy to crystal structures of related aptamers from other organisms 102 while also providing information on dynamics. RNA-protein complexes I had been interested for a while in studying a protein–RNA complex when one of my postdocs came back from a meeting where Philippe Bouvet from Toulouse, France had talked about nucleolin. The first two nucleolin RRMs (the most common single-stranded RNA binding motif, then called RBD) had been shown to bind to specific consensus sequences in pre-ribosomal RNA. 103 Fortunately, since I had no experience in protein NMR, postdoctoral fellow Frederic Allain had joined my group. He had solved the first NMR structure of a protein–RNA complex (U1A RRM with U1A mRNA internal loop) as a graduate student with Gabriele Varani. 104 In collaboration with Philippe, who did a sabbatical in my lab for 3 months in 1998, Fred solved the NMR structure of nucleolin RRM1–2 alone and with a target RNA hairpin. 105 , 106 He showed that the linker between the two RRMs only became structured on binding RNA (thus the two RRM structures could be solved separately). In the complex with RNA, the two RRMs bound on opposite sides of the structured hairpin loop, and one of the key (and cool) features of the structure was the insertion of the protein side chains of the linker K94 and RRM1 F56 into the RNA loop from opposite sides, thereby filling a hole in the loop. Later studies, including some from our lab, showed that RRMs can have structural variations and bind specific RNAs in many different ways. 107 In collaboration with my department colleague Guillaume Chanfreau, we investigated by NMR the RNA recognition by a double-strand RNA binding domain (dsRBD) found in S . cerevisiae (yeast) RNase III that targets the endonuclease domain to specific sites for cleavage. 108 Graduate student Haihong Wu determined the structural basis of the dsRBD recognition of a unique class of AGNN tetraloops, where N is any nucleotide. 109 , 110 The dsRBD interacts along one side of the helix, binding successive minor, major, and tetraloop minor grooves, where the insertion of an α-helix into the AGNN tetraloop minor groove determines its specificity. Prior to that, it was thought that dsRBDs bound double-stranded RNA without sequence specificity. This interaction was significant in explaining how the dsRBD positions the endonuclease domain of the RNase III 14–16 bp away from the tetraloop for target cleavage (processing) of the specific RNA sites. These studies in the early 2000′s were important as early examples of how RNA binding domains recognize RNA, of interest both to RNA biologists and NMR spectroscopists. But they attracted our interest in part because their size made them doable by NMR. It was time to delve more deeply into biology. 20+ years of telomerase, the RNP that maintains the 3′-ends of linear chromosomes When I turned 50, I decided that I was going to solve the structure of telomerase. Telomerase is an RNA–protein complex that maintains the 3′-ends of linear chromosomes. 111 , 112 Its minimal components for catalysis are telomerase reverse transcriptase (TERT) and telomerase RNA (TER) that contains a template that is used for processive synthesis of multiple telomere repeats (TTAGGG in human, TTGGGG in Tetrahymena); however, telomerase holoenzymes contain additional proteins, many of which had not been identified when I started this project. I had been interested in telomerase since my group first solved the NMR structures of G-quadruplexes formed by Oxytricha nova telomere repeats. 44 , 62 I thought that it was time to take on a challenging risky project. Fortunately I did not fully appreciate how challenging it would be. I was specifically interested in figuring out the role of the telomerase RNA (TER) beyond the template. The template is contained within a domain called the template/pseudoknot which together with another conserved domain (CR4/5 in human) are essential for TERT activity. 113 , 114 I had tried for several years to get someone to work on this, and finally postdoctoral fellow Carla Theimer took up the challenge. She had worked on thermodynamics of pseudoknots as a graduate student, so she combined her previous training with learning macromolecular NMR of RNA in my lab to identify the essential elements of the conserved pseudoknot in human TER (hTR). Carla’s structure of this minimal pseudoknot showed a series of base triples where the loops interacted with the stems 115 ( Figure 13 ). To my knowledge this was the first major groove triple helix found in RNA and was a surprise to many RNA biologists, who assumed that the RNA major groove was too narrow to accommodate a third strand, but seemed natural to me from our work on DNA triplexes. Our 2005 publication on the human telomerase pseudoknot is my fifth most highly cited paper. After our structure was published, many other major groove triple helices were discovered in aptamers, riboswitches, and other non-coding RNAs such as MALAT1. 116 , 117 Over the next decade we continued our structure–function studies of human telomerase RNA t/PK (core domain) and the CR4/5 RNA elements that are essential for TERT activity and also of elements of the H/ACA RNA domain, in collaboration with Tamas Kiss, important for TER biogenesis and localization. 118 , 119 In one study of particular significance to me, postdoctoral fellow Qi Zhang, together with postdoc Nak-Kyoon Kim, extended our NMR studies of the t/PK domain, to determine the unexpected structure, dynamics, and functional importance of a 5 nt bulge, conserved in length and location but not sequence, that causes a ~90° bend between the flanking helices and also determined a model of the full-length pseudoknot. 120 This work was published as my Inaugural Article in PNAS for my 2009 election to the National Academy of Sciences; remarkably Qi’s model fit the pseudoknot RNA almost perfectly in the first sub-4 Å cryo-EM structures of human telomerase published more than a decade later. 121 – 123 Figure 13. Open in a new tab Cover of the March 4, 2005 issue of Molecular Cell that our paper on the NMR structure of the conserved human telomerase RNA pseudoknot appeared in (reference 115 ). The base triples observed between the stems and loops are shown. The three blue–orange major groove triples became known as the triple helix. Shortly after we started investigating human TER (hTR), we also began studying Tetrahymena thermophila telomerase. We started with structure–function studies of TER. Soon after, we started collaborating with telomerase biochemist Kathy Collins, and she generously hosted me for a 6 month sabbatical in her lab in Berkeley (Sep 2006 – Mar 2007). There, I learned how to culture Tetrahymena and affinity purify Tetrahymena telomerase from strains where TERT had been tagged at the endogenous locus with a C-terminal 3XFlag-TEV-ZZ (as well as many other things). On February 16, 2007 I brought my purified Tetrahymena telomerase to Eva Nogales lab at Berkeley where her technician Patricia Grob collected the first negative stain images of telomerase. Shortly after that my sabbatical in UC Berkeley ended, and the EM project did not move forward until several years later. Meanwhile, postdoctoral fellow Mahavir Singh had been working with little success on domains of Tetrahymena telomerase assembly protein p65, that had been identified as a La-related protein 7 (LARP7). 124 In desperation, he deleted a 48 aa unstructured region from middle of the C-terminal domain and was able to solve its structure and complex with TER. 125 It turned out to be an unusual RRM, 126 the most common single-strand RNA binding domain, with an exceptionally long 48 residue β2–β3 loop! Later we found that human Larp7 RRM1 contains an even longer α2–β4 loop (189 aa). 127 With Kathy’s blessing we had resumed EM studies of Tetrahymena telomerase in collaboration with Prof. Hong Zhou at UCLA, and postdoc Jiansen Jiang joined the project in 2010. Kathy had meanwhile identified two additional Tetrahymena telomerase subunits (p82 (Teb1) and p50) and characterized them in part by making individual protein-tagged strains. 128 Using negative stain EM of Tetrahymena telomerase that we purified from 10 strains with different protein or subunit tags and labeled with the Fab-fragment of anti-Flag antibody (or MS2-coat protein for TER), Jiansen, working together with another postdoc Edward Miracco who did all the biochemistry, was able to localize all but one (p45) of the 7 known proteins (TERT, p65, p75, p45, p19, p82, p50) and fit the p65 RRM2–TER structure correctly in the negative stain EM map. Bosun Min from Collins lab did reconstitution assays to show that our subunit arrangement was correct. This study appeared in Nature (2013) 129 ( Figure 14A ). Figure 14. Open in a new tab Structures of Tetrahymena telomerase (A) Negative stain EM structure ~25 Å, with positions of subunits identified by Fab-labelling (2013), (B) 9 Å cryo-EM structure with all subunits identified (2015), (C) 4.8 Å cryo-EM structure with telomeric DNA (CST masked out) (2018), (D) 3.3 Å cryo-EM structure with telomeric DNA (CST masked out), the highest resolution structure of four at different steps of telomere repeat synthesis (2021), (E) 3.5 Å cryo-EM structure with first complete model of Tetrahymena CST (2022), (F) Model of cryo-EM structure with polymerase α-primase (2022), (G) cryo-EM structure of holoenzyme with first complete model of p65–TER (2023). 129 , 130 , 133 – 135 , 141 Two years later, we published the first cryo-EM structure of telomerase at 9 Å resolution 130 ( Figure 14B ). This is one of my favorite of our papers on telomerase because it was like putting all the pieces of a puzzle together. In addition to Jiansen’ and Edd Miracco’s cryo-EM structure, graduate student Henry Chan determined crystal structures of two domains of a heterotrimeric complex p75–p45–p19 that allowed us to propose it as an Replication Protein A related complex called CST (Ctc1–Stn1–Ten1); graduate student Darian Cash determined an NMR structure of the Tetrahymena telomerase RNA pseudoknot; mass spectrometry analysis with the Joe Loo lab allowed Henry to confirm the identification of two previously unknown subunits (Teb2 and Teb3) that formed another RPA related complex with Teb1; Heather Upton from Kathy Collins lab performed telomerase reconstitution activity assays with different subunits and amino acid substitutions that confirmed the importance of proposed interactions; and Jiansen identified where the telomeric DNA exits telomerase by linking two telomerase holoenzymes through streptavidin binding to biotin-5′-DNA primer and visualizing by negative stain EM. We were able to fit the cryo-EM map with known protein and RNA domain structures or homologous domain structures (e.g. RPA) to model all or part of the 10 subunits (TERT, TER, p65, Teb1, Teb2, Teb3, p75, p45, p19, p50) except p50. For example, there were crystal structures of TERT N-terminal TEN domain from Tetrahymena 131 and a TERT RBD-RT-CTE ‘ring’ from a minimal TERT from Tribolium casteneum (flour beetle). 132 Previously, it was thought that the Tetrahymena telomerase proteins besides TERT had no homologies to human telomerase proteins. However, based on this work we proposed that p50, Teb1, and p75–p45–p19, which constitutively bind Tetrahymena telomerase, were homologous to human TPP1, POT1, and CST that only transiently associate with telomerase. Thus, Tetrahymena telomerase turned out to be an ideal model system for studying telomerase and its interactions at telomeres. Three years later, Jiansen obtained a cryo-EM map of Tetrahymena telomerase with telomeric DNA at a resolution of 4.8 Å by masking out the dynamic p75–p45–p19 (CST) complex, Yaqiang Wang modeled TER, and Lukas Susac painstakingly traced a model of the complete TERT for the first time ( Figure 14C ). They discovered an unexpected complex between the TEN domain and an insertion in the TERT RT domain (named TRAP), and we showed that TEN–TRAP plays a critical role in telomerase activity. This paper was published in Cell , 2018. 133 And then, COVID came, with lockdowns ‘safer at home’ at UCLA starting March 20, 2020. Fortunately, we had a lot of data and time to analyze and write about it, and once we were able to go back to lab (in shifts) everyone was super motivated to work. So, the next couple of years turned out to be a very productive time in the lab –– we published three Nature papers on telomerase in 2021–2022. 121 , 134 , 135 Postdoctoral fellow Yao He, with help from Yaqiang Wang, Baocheng Liu, and other lab members obtained three more structures of Tetrahymena telomerase at different steps of telomere repeat synthesis, with the highest resolution structure at 3.3 Å ( Figure 14D ). So we had 4 structures, at steps 2, 3, 4, and 5, i.e. the number of nucleotides that would be added as the 6 nt telomere repeat was copied off the template. Finally, we could say something about mechanism! We could clearly see the number of template–DNA base pairs in the catalytic cavity, changes in the single-strand linkers on either side of the template that stretched and compacted like an accordion 136 to allow the template to move through the active site, anchor points for TER on TERT to prevent movement beyond the end of the template, and many other details. 134 For me, one of the most exciting results was finding that only 4 base pairs (or transiently 5 before moving out of the active site) were present at each step. Before that I thought the question was how to separate the template–DNA duplex so that the template could translocate back to the beginning for each round of telomere repeat synthesis, but with such a short duplex the question became, how does it stay together until it is time for template translocation? In all of our higher resolution cryo-EM structures of Tetrahymena telomerase, the putative CST (p75–p45–p19) complex had been masked out. We were finally able to determine its structure by combining the three new data sets and doing focused 3D classification with a spherical mask around the CST density 135 ( Figure 14E ). But to really know if it was functionally CST, we needed to study its interaction and activity with polymerase α-primase ( Figure 14F ). In humans, CST recruits and interacts with polymerase α-primase for synthesis of the C-strand that is complementary to the G-strand synthesized by telomerase. Happily, our paper on this work, spearheaded by Yao He and He Song, was published concurrently with three others on human CST–polymerase α-primase at different steps in the process. 135 , 137 – 139 Another thing that made me happy was that for this study we had to use both NMR data, obtained and analyzed by my graduate student Henry Chan and postdoc Lukas Susac, and cryo-EM data. It turned out that part of the interface between the Ctc1 (p75) and p50 was invisible in the cryo-EM map; we knew this because residues beyond what we could see had been shown to be essential for function 140 and Henry had shown that peptides from this region bound to Ctc1 OB-A by NMR, in work that was only published in his thesis (2017). One day I walked in the office and found Yao digging through Henry’s old NMR data, which finally got published in 2022 135 ! In another example of combining NMR and cryo-EM, we ended up using some of Mahavir Singh’s old unpublished NMR data, from his extensive studies of his difficult protein p65 almost a decade earlier, to identify sequences of some previously unknown helices we saw in a focused refinement of p65–TER in telomerase in cryo-EM maps. 141 As I like to say, NMR and cryo-EM are the perfect pair for structural biology of protein–nucleic acids complexes. Our structural biology studies of Tetrahymena telomerase provided many insights into human telomerase structure and allowed us to predict models for TEN–TRAP and TPP1 interactions. 142 Meanwhile, we had continued to work on human telomerase ( Figure 15 ). Kelly Nguyen had published an ~8 Å cryo-EM structure of human telomerase in 2018 while a postdoc, 143 and in 2021–2022 the Nguyen, Lei, and my lab ( Figure 15 ) all published sub-4Å structures. 121 – 123 Both my lab, largely through the efforts of Baocheng Liu and Yao He, and the Nguyen lab solved human telomerase structures with shelterin protein TPP1 bound (and also POT1 for Nguyen). 121 , 144 We were able to see the detailed three-way interface between TPP1–TEN–TRAP that recruits telomerase to telomeres, we confirmed that the structural and functional homologies between human and Tetrahymena telomerase include a four base pair duplex ( Figure 15 ), and we mapped numerous disease mutations to the TERT–TER interface that highlighted the importance of TER for telomerase assembly and activity. Finally, over the course of almost two decades, we had achieved the goal that I set for myself when I turned 50 ( Figure 16 ). Which is not to say we are done with telomerase! Figure 15. Open in a new tab Structure of human telomerase with shelterin protein TPP1 and comparison to Tetrahymena telomerase. (A) Schematic of human telomerase RNA (TER). Gray regions are low resolution or invisible in the cryo-EM map. (B) Cryo-EM structure of telomerase catalytic core RNP of TERT, TER, and H2A/H2B dimer at 3.3–3.5 Å and H/ACA RNP with Dyskerin, Gar1, Nhp2, Nop10, and TCAB1 at 3.2 Å resolution. TERT domains are colored as in C-E. (C-D) Comparison of TERT–TER catalytic core and TER–DNA for human (C) and Tetrahymena (D) telomerase, illustrating their similarity. Both have similar TERT domains and template with single-strand linkers in the catalytic cavity, and four template–DNA base pairs at the second step of telomere repeat synthesis. (E) Schematic of domains of TERT and TPP1, colored as in the structures, and interaction regions between TPP1 and TERT. 121 , 134 , 155 Figure 16. Open in a new tab August 19, 2022, first Feigon lab barbeque since Covid (the only two years Juli did not have a summer party at her house since starting at UCLA). We had a lot to celebrate! (and lots of delicious food from the farmer’s market to eat). 7SK RNP, a major regulator of eukaryotic transcription 7SK RNA is a ~332 nt non-coding RNA that plays an important role in regulating RNA Polymerase II (RNAP II) transcription elongation by binding and inactivating P-TEFb (positive transcription elongation factor b) in a 7SK RNP complex. 145 , 146 Kinase activity of P-TEFb is required for transition of promotor proximal paused RNAP II to productive elongation and it is also an essential host cofactor for HIV-1 viral replication. 147 I got interested in 7SK RNP initially through our studies of Tetrahymena telomerase assembly protein p65. I had suggested to Catherine (Katie) Eichhorn that for her postdoctoral fellowship applications she propose to study the role of human Larp7, that binds 7SK RNA, in 7SK RNP assembly. Katie enthusiastically took up this challenge, and ended up combining NMR and X-ray crystallography to determine the structural basis for Larp7 recognition of the 3′-end of 7SK RNA. 148 Then postdoc Yuan Yang joined the project, and working with Katie she solved crystal structures of MePCE (methylphosphate capping enzyme) bound to the 5′-end of 7SK RNA in the presence of SAH pre- and post-catalysis, 149 revealing the mechanism of capping. MePCE uniquely adds a γ-methyl to the 5′-phosphate of 7SK, so this project was my first foray into methylases. Fortunately, I had Yuan as well as my colleague Steve Clarke as guides. Structural biology of 7SK RNP is a different and even more difficult kind of challenge from telomerase and folded RNAs like riboswitches––7SK does not form a single RNP, but rather binds different proteins depending on whether it is sequestering P-TEFb or not, 150 and the RNA itself is highly flexible with only local regions well defined. Furthermore 7SK RNA adopts at least two different conformations depending on whether P-TEFb is bound or released. 151 Despite these challenges, Yuan and another postdoc Shiheng Liu have succeeded in determining cryo-EM structures of MePCE–7SK–Larp7 core RNP (with minimal RNAs) that explain how the 7SK RNA can change conformation while constitutively bound to these two proteins, why MePCE remains bound but is catalytically incompetent, and the 7SK RNA dependence of MePCE–Larp7 interaction in the 7SK RNP. 127 And our collaborators Sylvain Egloff and Tamas Kiss heroically worked during Covid to provide cellular studies that supported our conformational switch model. Forty years after the discovery of 7SK RNA and twenty five years after the identification of a role for 7SK RNA in transcription, 152 , 153 we have the first structural information on 7SK RNP and its regulation. There is still a lot to do! Reflections and Advice Be as critical of your collaborator’s work as you are of your own. Let your competitors motivate you. Some of my most important and creative papers were published in JMB . It is important to go to different labs and different schools during your training; it broadens your perspective on how science is done. It is also important because it exposes you to different mentoring styles, so you can find what suits you. Always be willing to abandon a hypothesis when it turns out to be wrong. One of the most wonderful things about doing science is becoming part of an international community. Be careful in judging your colleague’s past actions by the societal norms of today. Science is hard. Be careful not to ascribe all the challenges you face to bias against you for your gender, race, religion, etc. It is better to be correct than first. To graduate students: Choose a lab where the science is your passion and the mentor is someone you can work with. If you choose a lab based on a research area you think you can get a job in, the best thing that can happen is you will get a job in a field that you don’t love and the worst thing is that you won’t be able to get a job in that field anyway. What has changed for women in science since I started: Women scientists no longer have to be 10X better than their male colleagues to succeed. Women faculty members are no longer advised to wait until they get tenure to have kids. What one woman scientist does or says does not represent all women scientists. Overview and future directions I have been lucky to have lived a life of intellectual challenge and science, and especially to have worked with so many brilliant and wonderful people. I have been fortunate to have become part of an international community of science that has brought me friendships from all over the world. I have attended meetings in so many beautiful and interesting places that enhanced my life while also advancing my science. I have also been privileged to have done science during a time when, at least initially, it was easy to get grant funding, and the US was a place that the brightest minds wanted to come to enhance their careers. Many who came here, in the first half of my career mostly from Europe and later many from Asia, came with the expectation that they would return to their home countries, but ended up wanting to stay in the US, thus enhancing US science. Others that did return brought the scientific and societal values they learned in the US home, thus moving science forward in that way. I don’t know where my place in science will be 10 years from now, or more importantly whether the luck of my generation of scientists will continue to the next generation. For pictures of all the structures that I have discussed in this retrospective, and more, please see the FeigonLabStructuresPlayingCards on our webpage. Our webpage also contains information on my small effort to help graduate students, postdocs, and research staff who are primary care givers of dependent children or parents by providing a small award that can be used for anything to relieve their domestic burden JF Helping Hands Award . Acknowledgements Above all, I would like to thank all of my group members, both those whose work I was able to discuss in this retrospective and those I could not, you are my scientific legacy. I gratefully acknowledge my PhD mentor David Kearns, postdoc mentor Alex Rich and unofficial postdoc mentors Andrew H-J Wang and Bob Griffin and the important contributions and friendship of collaborators Bill Denny, Jacques van Boom, Gijs van der Marel, Gerry Harbison, Vladi Sklenar, Jim Roe, Peter Dervan, Dave Bartel, Jack Szostak, Larry Gold, Todd Yeates, David Eisenberg, Richard Lehrer, Frank Anet, Danny Neuhauser, Irvin Chen, Reid Johnson, Stefan Grzesiek, Philippe Bouvet, Guillaume Chanfreau, Wayne Hubbell, Peter Qin, Ite Laird-Offringa, Michele Caizergues-Ferrer, Kathy Collins, Rob Clubb, Tamas Kiss, Sylvain Egloff, Michael Stone, Julian Chen, Chris Switzer, Hong Zhou, Yehuda (Dudy) Tzfati, Shu-ou Shan, Duilio Cascio, Joe Loo, Joe Yesselman, Marcus Gallagher-Jones, Ryan Palumbo and Steven Hanes; I also had fun writing editorial reviews with David Lilley, Carlos Bustamante, and a meeting review with Olke Uhlenbeck and Art Pardi for the first ever RNA structure meeting. My heartfelt gratitude goes to Vladi Sklenar for helping make my success in my early career possible and Kathy Collins and Hong Zhou for helping me achieve my dream of solving the structure of telomerase. I thank my friends in the NMR, structural biology, and RNA fields for their support and friendship including (but not limited to) Kees Hilbers, Angela Gronenborn, Art Palmer, Ann McDermott, Tatyana Polenova, Hashim Al-Hashimi, Michael Summers, Ad Bax, David Wemmer, Christian Greisinger, Harold Schwalbe, Teresa Carlomagno, Mitsu Ikura, Matsasune (Kai) Kainosho, John Markley, Gerhard Wagner, John Kuriyan, Tom Cech, Peter Wright, Olke Uhlenbeck, Nacho Tinoco, Helen Berman, Jennifer Doudna, Elizabeth Blackburn. I sadly acknowledge departmental colleagues who have passed away, David Sigman, Dan Kivelson, Francois Diederich, and Chris Foote, all of who showed me an unusual level of respect and kindness while I was finding my way to a career, and I thank current and former female colleagues in my department Sabeeha Merchant, Carla Koehler, Andrea Liu, Cathy Clarke, and Joan Valentine for their support and insights. Research in the Feigon laboratory is currently supported by NIH grants R35GM131901 and R01AI155170. Other funders through much of my career include NSF and DOE. Footnotes DECLARATION OF COMPETING INTEREST The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References 1. Suddath FL, Quigley GJ, McPherson A, Sneden D, Kim JJ, Kim SH, Rich A, (1974). Three-dimensional structure of yeast phenylalanine transfer RNA at 3.0angstroms resolution. Nature 248, 20–24. 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