By Ribosome Possessed - Journal of Biological Chemistryslicked-back wavy black hair, a spotless white lab coat, stylishglasses,andimmaculatelypolishedshoes.Shyam chain-smokedashemonitoredthemovementoftheblaz-

By Ribosome PossessedPublished, JBC Papers in Press, June 27, 2013, DOI 10.1074/jbc.X113.497511

Harry F. Noller

From the Center for Molecular Biology of RNA, Department of Molecular, Cell andDevelopmental Biology, University of California, Santa Cruz, California 95064

I’ll play it, and tell you what it is later.—Miles Davis

“Listen, howwould you like to come on tourwithmynewband?” BennyWilson askedme in the summer of 1965 duringmy final year of graduate school. A few days later,I received a letter from the National Institutes of Health (NIH) with the news thatI had been awarded a three-year fellowship to do postdoctoral research at the

Medical Research Council (MRC) Laboratory of Molecular Biology in Cambridge. Benny was anexciting young jazz pianist and blues organist, the son of a Baptist minister in Eugene who trans-ported his Hammond organ to gigs in the back of his elegant blueMercedes 220S Cabriolet. It wasa great temptation to join Benny on the road, but there would be scant opportunity to do bio-chemistry. On the other hand, I guessed that I would findmusicians in Cambridge. That fall, in ourfinal weekend in the Bay Area, my wife and I found ourselves at Ken Kesey’s home in La Honda,where his famous bus “Furthur” was parked in the driveway. We watched hours and hours of raw16-mm movie footage taken by the Merry Pranksters on their infamous trip, surrounded byhippies and Hells Angels. In the morning, we raced up La Honda Road to Skyline, where we hadbreakfast with Neal Cassady, at the counter of Alice’s Restaurant in Woodside. Cassady was asvivid as Kerouac had portrayed him as DeanMoriarty inOn the Road.At breakfast, hemaintaineda rapid streamof incomprehensible parallel conversations: withme, the person sitting on the otherside of him, the waitress, and someone who was not there. Over the next few days, we reluctantlysaid goodbye to our native culture, crossed the country to New York by train, and boarded acut-rate Icelandic Airlines propeller flight to London via Keflavik.My postdoctoral adviser, theWelsh protein chemist Ieuan Harris, had booked us a room at the

Prince Regent, a nice pub near the middle of Cambridge. On our first morning, I rolled out of bed,took the bus to theMRC, and was greeted by the receptionist with “Oh, Dr. Noller. Let me go findDr. Perutz!” I was stunned. Did she mean Nobel laureate Max Perutz, director of the MRC Labo-ratory ofMolecular Biology and one of the gods ofmolecular biology? I followed her down the hall.She said, “Dr. Perutz is out of his office. Let’s see if he’s in his laboratory.”Just then, the door of the cold room flew open, and a small baldman in a white coat stepped out,

carrying an Erlenmeyer flask containing a red liquid.Max Perutz, founder of the science of proteincrystallography, was doing a hemoglobin prep with his own hands. He stopped, smiled modestly,extended a free hand, and welcomed me warmly to the MRC. I was struck by his utter lack ofpretention and his generosity in taking time to talk with an obscure postdoctoral fellow on his firstday, even as condensation formed on the surface of his Erlenmeyer. I felt very out of place, in wayover my head, and very lucky.

Wat is de Bedoeling van het Leven?—Jack Moore

I was born in Oakland and grew up in the East Bay. Neither of my parents had been to college,but they andmy grandparents stressed the importance of education. They encouraged and pushedme every step of the way as best they could.My father, Harry F. Noller, Sr., the first-generation sonof an Englishman and a Swede, was a self-taught mechanical engineer with a high school educa-

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 34, pp. 24872–24885, August 23, 2013© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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tion. During his career, he made the unlikely transitionfrom a job as draftsman to leading the team of engineersthat developed the first mechanical calculator with amemory at Marchant Calculating Machine Co. inEmeryville. My mother, Charlotte Silva Noller, whosefamily were Portuguese from theAzores, had gone to busi-ness school and became a full-time homemaker after I wasborn. After World War II, we lived in a one-bedroomapartment in Berkeley on lower Dwight Way, surroundedby a vast industrial slum. When the wind came from thesouth, we inhaled pungent aromas emitted by the tall Cut-ter Laboratories smokestack across the street. What wasgoing on in those “laboratories,” I wondered? I was one ofthe few Caucasian kids at Columbus School, where I hadmany friends and supportive teachers. In a well intendedbut misguided moment, my teachers advised my parentsthat I should skip a grade. I was interested in everything,particularly things to dowith “science,” including the LoneRanger Atom Bomb Ring that I sent away for with someKix breakfast cereal box tops. On it was mounted a tinytelescope-like viewer, inside of which you could watch thetracks of subatomic particles as they were emitted fromsome unspecified radioisotope.Following the war, the atomic bomb was always in the

news. From the sidewalk in front of our apartment,my dadpointed out a white circular structure that was just visibleover the eucalyptus trees, high in the Berkeley Hills. Thestructure housed the cyclotron, he explained, where scien-tists had “split the atom” and helped to develop the atomicbomb. Little couldwe imagine that fifty years later, I wouldbeworking in that very building collecting x-ray data usingsynchrotron radiation. Our neighbor Dick Lukens was abiochemist. He listened patiently to my questions, evenloaning me a sophisticated college paleontology textbookwhen I asked about dinosaurs. My dad took me to ChabotObservatory in the Oakland Hills, where the public couldlook at the planets and stellar objects through a powerfultelescope. Sometimes he took me with him to Marchant,where everyone greeted him with a smile, from the screw-machine operators and secretaries to the engineers. I wasfascinated by the enormous complexity of the insides ofthe calculators, with over 2000 rapidly moving parts thatinteracted precisely with one another to add, subtract,multiply, and divide, whirring and clattering as they pro-cessed their mechanical arithmetic. My dad told storiesabout a man he knew named Dr. Cornish, a sort of inde-pendent medical researcher who was in the headlines forbringing a dog called Lazarus “back to life” in his lab inWest Berkeley. Many interesting things were happening

right around me. At the age of 6 or 7, I began to wonder,how do youmake a living as a scientist? It was not obvious.My parents built a modest home in Orinda, just to the

east of the Berkeley Hills, where we moved in 1948. With-out a doubt, we were the least affluent family in thatupscale community. In the sixth grade at the OrindaUnion School, I was lucky to have, asmy teacher, the won-derful Verna Givens, who sacrificed a major chunk of hersalary to buy a high-quality microscope for our class. Shelimited its use to only a few selected students, and I waslucky to be one of them. With that microscope, I watchedprotozoa swim around in pond water for the first time. Inthe eighth grade, much to the detriment of our academicperformance, my friends and I discovered the intriguingand weird world of science fiction magazines, where wewere introduced to many far-out concepts, some real andsome imaginary, by the pioneering writings of RobertHeinlein, Philip K. Dick, Arthur C. Clarke, Ray Bradbury,and others. We speculated about “the fourth dimension,”time travel, and relativity. We had no idea what we weretalking about, but we were getting very high on an ava-lanche of strange new ideas.In 1952, I entered Acalanes High School, an institution

blessed with an extraordinary faculty, many of whom hadbeen educated at the University of California, Berkeley,bringing its high standards along with them. Some, likeJosephine Ochoa, our intimidating Latin teacher, instilledin us academic discipline and fear that brought the topstudents in our class to their knees. Our exceptional phys-ics teacher, John Annis, introduced us to the concepts ofexperimental controls and intellectual rigor. However, Iwas most intrigued by biology and chemistry. Berkeleyscientists were again making the headlines, but this timethey were not the Manhattan Project physicists. Now, theheadlines of the Oakland Tribune announced “Life in theTest Tube” and “Secret of Life” in stories about the recon-stitution of tobacco mosaic virus (TMV) from its purifiedprotein and RNA inWendell Stanley’s lab and the demon-stration that the purified TMV RNA by itself could causetobacco plants to produce the virus. When the list ofmajors offered at Berkeley was passed around our highschool class, recalling our family friend Dick Lukens, theword “biochemistry” popped out at me. Did this meanwhat I thought it did? Could it be possible that you mightbe able to do both chemistry and biology? For an entirecareer? One day, I decided to drive into Berkeley to findout for myself. The biochemistry department was housedin a bland four-story concrete building that stood acrossthe road from the Cal football stadium. Over the entrancewere the words “Biochemistry and Virus Laboratory.” I

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was thrilled. “Virus Laboratory!” Magical words. On thewalls of the lobby were huge blowups of beautiful electronmicrographs of viruses: bacteriophage T2, poliovirus, andright out of the headlines, the famous TMV, taken by thepioneering electron microscopist Robley Williams. Strug-gling to containmy excitement, I told the receptionist thatI wanted to find out about biochemistry. She picked up thephone and called Professor DonaldMcDonald, a carbohy-drate chemist. He welcomed me into the office next to hislaboratory and spent about an hour patiently telling meabout biochemistry, what his research was about, andwhat the biochemistry major at Berkeley was like. Profes-sor McDonald’s generosity that day, in taking time to talkto a high school kid who walked in off the street unan-nounced, had a huge impact on me.My parents could not afford to sendme away to college,

and because Berkeley was a fifteen-minute drive from ourhouse, it was a foregone conclusion that I would go tocollege at Cal. My mother’s father, M. R. Silva, who hadhimself quit school in the fifth grade to take a job packingbullets at the Hercules Powder Co. to help support hisfamily, stepped in and sprung for the $54 per semesterfees, plus the cost of my textbooks. I began commutingfrom Orinda to Berkeley, drifting through the corners ofthe twisty Wildcat Canyon Road in my MG TD. After therigor of Acalanes, the classes at Berkeley did not seem allthat difficult. The exception was calculus, which wastaught completely and incomprehensibly by teachingassistants; I nearly ended my career, along with my sanity,in trying to understand the formal definition of a limit aspresented in our impenetrable calculus textbook. To fulfillthe biochemistry major requirements, which includednearly all of the classes required for both the chemistry andbiology majors, I was often in class from 8 to 5.On top of this, I was playing the saxophone with various

bands in the evenings. On the weekends, I worked withfriends who were building and racing sports cars andmotorcycles. I rode my 500cc BSA Gold Star on the trailsin the Oakland Hills. A girlfriend studying in the theaterdepartment recruited me to play in the pit band for theMask and Dagger Review and on stage for a production ofGeorg Buchner’s Woyzeck. On Thursday nights, therewere the legendary jam sessions at George’s NorthgateCafe on Euclid Avenue near the campus, where I sat inwith some of the best musicians in the Bay Area whilepuzzling over physical chemistry problem sets at a backtable between tunes. Many of those players at Northgatehave since earned their own bins at record stores, includ-ing the saxophonist Pharoah Sanders, the bassist BarrePhillips, and the pianist-composer Clare Fischer. Our

usual drummer was the (now) film and Broadway actorLarry Bryggman. Along with themusic scene, I discoveredthe Berkeley hipster underground, where I met many ofthe characters who had migrated to Telegraph Avenuefrom North Beach: people with names like “BarneyGoogle,” “Hube theCube,” and “Leonard the Locomotive.”I was getting spread dangerously thin.After graduating from Berkeley in 1960, my first

research opportunity was a year working as a lab techni-cian in the Laboratory of Medical Entomology at the Kai-ser Foundation Research Institute under Eliezer Benja-mini, an entomologist studying flea-bite hypersensitivity. Iwas first stationed on the grounds of the United Statesgovernment plague lab at the Presidio in San Francisco,where we collected and analyzed flea saliva. At lunch, I gotmy first close-up exposure to seasoned scientists, theircontests of wrywit interlacedwith spellbinding anecdotes:the suave Leo Kartman’s stories about tracking down thecausative agents of tropical diseases, which led to hisknighthood, and Bruce Hudson’s accounts of trappingplague-infested rodents in the High Sierras. On onemem-orable excursion, we visited an abandoned house in theFillmore District, where police had discovered a corpsealong with an infestation of human fleas, with which wehoped to replenish the lab’s dwindling Pulex irritans col-ony.We wore white pants fastened at the ankles with rub-ber bands and entered the house armed with hand-heldaspirators. Minutes into our safari, my pants seemed tohave become spray-painted gray around the ankles, amotif that slowly moved up my pant legs as we maneu-vered around the blood-stained bed. We worked quickly,recovering a large jar full of P. irritans. Even after shower-ing thoroughly at the lab and decontaminating our clothesin a creosote-laced tub, a few dozen fleas managed to sur-vive the trip home, much to my mother’s profoundanguish.In early 1961, I was moved to a lab at the Kaiser Foun-

dation Hospital in Richmond, across the Bay, where Ibegan working with the biochemist Janis Young, who hadearned her Ph.D. degree at Berkeley working on the aminoacid sequence of the coat protein of none other than TMVwith Heinz Fraenkel-Conrat. Janis was a talented bio-chemist and a patient teacher, generous enough to givemea free hand in much of our work. I was soon joined by anew co-worker at the Richmond lab who had just receivedamaster’s degree in entomology at Berkeley. He had spell-binding stories to tell: escaping from Russia to Germanyfrom the Soviet Union during the war, joining the HitlerJugend, jumping from planes with the U.S. 82nd AirborneDivision, doing hard time in the Maryland State Peniten-




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tiary for armed assault, and expulsion from the doctoralprogram at Berkeley after brandishing a loaded pistol on adepartmental field trip. My education continued toexpand in many dimensions.One evening, I even got to work in the legendary Fraen-

kel-Conrat lab, using their rotary evaporator to concen-trate a flaskfull of flea extract. The only other personwork-ing late was the distinguished Japanese protein chemistAkira Tsugita, a major player on the TMV project. He wassurprisingly friendly and showed me how to use the evap-orator, asked questions, and cracked jokes in his barelycomprehensible English, smiling through gold teeth. Thelab was small and cramped, equipment jammed in its nar-row aisles. It reeked of pyridine, the characteristic smell ofprotein chemistry in that era. It was hard to believe thatfrom these cramped and cluttered spaces came the magicthat had led to the discoveries of the “secret of life” and “lifein a test tube.” That evening, the untrained observermighteven have mistaken me in my white lab coat for one ofthose very researchers. My chest swelled with pride as theflea extract slowly reduced in volume under my steady,expert gaze.The end of my rookie year in a real lab was signaled by a

telegram with the message that I had been accepted tograduate school in the chemistry department at the Uni-versity of Oregon in Eugene with a teaching assistantshipto cover my tuition and living expenses. My immediatereaction was that someone must have made a huge mis-take. I was pleasantly stunned. This was another happyaccident, the outcome of which was not at all obvious tome at that moment.After my first year of graduate school in Eugene, which

was packed with courses, exams, and playing gigs with alocal group called The Jazz Prophets, I was accepted intothe laboratory of Sidney Bernhard at the Institute ofMolecular Biology. He had recently arrived from the NIH,where he had made his reputation in the field of enzymemechanisms, in particular, the serine proteases. Sidneyhad done his graduate work at Columbia with Louis Ham-mett, the father of physical organic chemistry. The Insti-tute of Molecular Biology’s atmosphere was much morelike that of an artist’s studio than a factory or hospital.Faculty and students clustered around the blackboard inthe hallway in open-ended discussions. On nice days, thewhole institute would go for potluck picnics. Sidney him-self played some jazz piano with me at parties. The sculp-tor LouisDurchanekwas our facilitiesmanager. Therewasprobably more scientific discussion over coffee (and ciga-rettes) than actual pipetting. It was a culture that nurtured(and respected) creativity of all kinds, including the scien-

tific kind. My project was to map the active site of theserine protease subtilisin using a novel substrate analogcalled furylacryloylimidazole. I made a stable furylacryloyl-subtilisin adduct, which we characterized kinetically andspectroscopically, and then used it to identify the sequencearound the active-site serine. While I was writing up thesequence paper, I received a wake-up call in the form of areport in Nature from Fred Sanger, who had scooped me.Nevertheless, I had enough results to write my Ph.D. the-sis. Sidney suggested that I go to Cambridge for my post-doctoral research to work with Ieuan Harris, who wasstudying a more challenging metabolic enzyme, a step ortwo up in complexity from serine proteases.

Who are you? What are you working on?—Sydney Brenner

My postdoctoral project in Ieuan Harris’s lab was todetermine the amino acid sequence of the enzyme glycer-aldehyde-3-phosphate dehydrogenase. At that time, theenzyme was the largest protein to be sequenced by usingtraditional direct protein sequencing methods. I collabo-rated with another postdoctoral fellow, the very talentedand funny Australian Barrie Davidson. Besides us, therewere two “research students” working on their doctoralprojects. One of them was Graham Jones, a red-headedWelshman who resonated with Barrie’s humor; the otherwas Jo Butler, a more traditional Cambridge student. Bar-rie and I got on well together and soon had a strong starton the sequence. Our use of ion-exchange columns to sep-arate the tryptic and chymotryptic peptides, a la StanfordMoore andWilliam Stein, was not received well by Ieuan.He tried to persuade us to use just a Sephadex sizingcolumn followed by paper electrophoresis, a simplerapproach that he himself had developed. Our use of highconcentrations of urea on columns, in an attempt torecover some of the poorly soluble peptides, came undermore serious fire when Ieuan discovered a cluster of ureacrystals clogging the flow cell of his precious UV detectorand spilling over onto the floor. While cleaning up themess and with Ieuan safely out of earshot, Barrie and Gra-ham burst into song, set to the melody of Leonard Bern-stein’s “Maria” fromWest Side Story.

U-re-a!I just made eight molar u-re-a!And, suddenly I found,My H-bonds came unbound�

Across the hall, Shyam Dube, a postdoctoral fellowworking with Brian Clarke and Kjeld Marcker, wassequencing methionyl-tRNA using the RNA oligonucleo-tide sequencing methods that Fred Sanger had developedjust down the hall from us. Shyam was a dapper guy, with




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slicked-back wavy black hair, a spotless white lab coat,stylish glasses, and immaculately polished shoes. Shyamchain-smoked as he monitored the movement of the blaz-ingly radioactive 32P-labeled tRNA through his column bythe buzzing of his Geiger counter, without gloves or radi-ation shield, his cigarettes burning a series of parallelbrown grooves in the wooden bench top. Shyam wasknown to disappear in themiddle of the afternoon, drivinghis large American sedan into the middle of Cambridge togo “tea dancing,” where he refined his skills at picking upattractive widows and divorcees. At an adjacent bench,Cesar Milstein, an Argentine postdoctoral fellow whowould later become known for his discovery of monoclo-nal antibodies, was trying to sequence an immunoglobulinlight chain using myeloma cell lines that overproducedsingle antibodies. Downstairs in the basement, John Abel-son,HowardGoodman, andArt Landy, threemoreAmer-ican postdoc fellows, worked night and day isolating andsequencing the su3 suppressor tRNA using the Sanger oli-gonucleotide method. Further into the basement, PaulSigler, Brian Matthews, and David Blow were hunchedover a small table wedged into a hallway connecting theMRC lab to Addenbrooke’s Hospital, building the firstmolecular model of chymotrypsin, adjusting the metalKendrew skeletal models of the amino acids with Allenwrenches and rulers, and measuring their positions froman electron densitymap displayed in a stack of transparentplastic sheets.With little warning, I had been dropped intothe very center of the rapidly exploding new field ofmolec-ular biology.In Berkeley, I had met Tony Tanner, who was a British

hipster on leave from Cambridge University and was tobecome Head of English Studies at King’s College. Tonyurged me to contact him when I arrived in Cambridge. Ifound him in his “rooms,” a sort of spacious, high-ceil-inged, chilly apartment in the ancient stone buildingswhere fellows of King’s resided. He invited us to a “sherryparty” that hewas giving the followingweek.We arrived tofind Tony’s rooms filled with tweedy Cambridge intellec-tuals: professors, dons, fellows, research students, andundergraduates. I felt very uncomfortable, partly because Idid not recognize a soul in the room apart from Tony andpartly because they all seemed at least an order of magni-tude brighter and more articulate than anyone I had evermet. I quickly found a glass of sherry and tried to avoidexposing my 200-word Californian vocabulary by stayingas inconspicuous as possible. As my anxiety peaked, I wasapproached by aman of compact dimensions, whose pres-ence dominated the room. I immediately recognized himfrom his distinctive South African accent and from his

visit to Eugene a few years before. “Hello. I’m SydneyBrenner. Who are you?” he asked. When he realized I wasworking at the MRC, he asked, “What are you workingon?” When I replied, he announced, “That’s stupid! Ifyou’re a protein chemist, why don’t you work on some-thing interesting, like ribosomes?” I felt catapulted into mynightmare of nightmares. Perhaps the most brilliant of allof the Cambridge scientists, the intellect most feared bythe lunch table of Nobel laureates at the MRC, whoselandmark research papers were the most subtle, elegant,and impenetrable, had singledme out in the crowd, expos-ing my utter scientific cluelessness to the Cambridge aca-demic society and thus to the world.In the following days and weeks, as I tried to retrieve the

fragments of my shattered ego, I found myself unable toget the word “ribosome” out ofmy head.What Sydney haddone, as he had done for many other young scientists, wasto try tomakeme realize that I have only one life to live andone scientific career. You can spend your life and careerworking on something boring or something exciting.What I had failed to realize was that the choice was up tome, and Sydney had forcefully pointed this out. I went tothe library at the MRC and began reading everything Icould find about ribosomes. I asked the other researcherswhat they knew about ribosomes andwhowas working onthem. Someone said that Alfred Tissieres was forming agroup in Geneva to study ribosomes. My next lucky breakwas when Ieuan told me that the Hungarian chemistMichael Sajgo would be coming to his lab and that hewould be replacing me. I wrote to Alfred to ask if he coulduse a protein chemist to work on ribosomes. He wroteback immediately, wanting to know when I could start.In the spring of 1966, my wife and I sold our MG YB

saloon (for £30), bought a Bedford van (for £54), installed amattress in the back of the van, and prepared to head forGeneva to meet Alfred. As we were packing, our landladyadvised us about survival on the continent. “Be sure topack a tinned ham,” she warned, in all seriousness. Westopped to visit friends in Paris, where we slept in the vanin a cushy neighborhood on the Ile Saint-Louis, and thendrove to Geneva via the Burgundian city of Dijon, wherewewere served a truly consciousness-altering coq au vin ata roadside restaurant. Alfred turned out to be utterlydelightful, with a civilized, almost aristocratic demeanorand a wry sense of humor. He was tall and gaunt, withwispy gray hair, deep-set blue eyes, and hollow cheeks. Itwas difficult to tell how old he was. He was from the can-ton of Valais in the Swiss Alps and an accomplished alpin-iste, who had participated in one of the first major Britishexpeditions to the Himalayas. He had been a graduate stu-




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dent in the biochemistry department at Cambridge, wherehe had driven a classic Bentley. He and the Bentley nextmoved to JimWatson’s lab at Harvard University to workon ribosomes. In Jim’s lab, Alfred had discovered that ribo-somes separated into two subunits when the magnesiumion concentration was lowered. Alfred’s own lab was nowattempting to characterize the protein composition of theribosome, so he wanted a protein chemist to come join inthe effort. I accepted on the spot. We returned to Cam-bridge via Monte Carlo. We watched the Grand Prix ofMonaco as John Frankenheimer was filming the movieGrand Prix, starring James Garner, with appearances bythe actual Formula 1 drivers. Most impressive was thecamera car, a powerful Lola T70 sports-racing car drivenby former Formula 1 champion Phil Hill, who drove thecircuit at speed, surrounded by the race cars, with an enor-mous 70-mmMitchell Panavision Reflex cameramountedjust behind his head.Alfred’s lab was populated by several of Jim Watson’s

students from Harvard, including Peter Moore, RayGesteland, and Gary Gussin. The founding postdoctoralfellow in his groupwas another American, RobTraut, whohad been introduced to protein synthesis as a graduatestudent in Fritz Lipmann’s lab at The Rockefeller Institute.Rob had then gone to the MRC, where he had done land-mark experiments on peptidyl transferase with RobinMonro. Rob was running polyacrylamide tube gels of 30Sand 50S subunits, trying to figure out just how many pro-teins there were. The gels he showedme were frightening:there were literally dozens of different protein bands.Worse yet, these proteins were impossible to work withbecause they were insoluble! To deal with this, PeterMoore was developing a column chromatography proce-dure, whichwas based on an earlier attempt by Jean-PierreWaller and which used a carboxymethylcellulose columnrun in 6 M urea with a linear salt gradient to separate theproteins of the 30S subunit. The flatter he ran the gradient,the more peaks appeared. Hajo Delius joined the lab fromPeter Hans Hofschneider’s laboratory at the Max PlanckInstitute for Biochemistry inMunich, and he began carry-ing out a similar approach for purification of the 50S sub-unit proteins.When I joinedAlfred’s lab, I began analyzingsome of the proteins that Rob and Peter had isolated. Wesoon confirmed our worst fears: the numerous gel bandsindeed corresponded to different proteins. The ribosomewas a lot more complicated than the simple virus-likerepeating-subunit structure that Jim Watson and othershad originally hoped for.Soon after arriving in Geneva in November 1966, we

attended the Geneva Jazz Festival, where we heard and

met the Jurg Lenggenhager Quartet, a group of youngavant-garde Swiss musicians, including Jurg on piano,Roger Pfund on bass (both from Bern), and AndreasStraub (from Basel) on drums. We were invited to a jamsession that night in a chateau in Nyon, a village along thenorth shore of the lake. We played until dawn. Theyinvited us to spend the summer with them in the South ofFrance at a pottery in the village of Pegomas, in the foot-hills between Grasse and Cannes. We played in galleriesand private parties around Cannes and Saint-Paul-de-Vence that summer and later in the Tobeco recordingstudios near the Zytgloggeplatz in Bern. I continued play-ing concerts around Europe with them during the rest ofmy postdoctoral stay in Geneva and then again in themid-1970s when I returned to Europe on sabbatical. The mid-1960s were a time of great social change, which was spill-ing over from the United States into Europe. The LivingTheater, headed by Julian Beck and Judith Malina, camefrom New York to Avignon, where we found them livingand rehearsing in an abandoned cloister. They were orbitedby a free-form circus-like troupe called The Human Family,founded by Oklahoman Jack Moore. We got to know manyof these people as they came through Geneva, performingtheir revolutionary theater to the astonishment and discom-fort of the conservative Swiss audiences.

I’ll bet you tried a lot of things that didn’t work.—Fred Sanger

In the spring of 1968, after two years in Geneva, Iapplied for an academic job in theUnited States.My lettersled to eight interviews and six offers. I worked my wayfrom the East Coast to the West Coast. My first interviewwas in the South Bronx, where an abandoned car missingits wheels, sitting on a carpet of broken glass, was visiblefrom the office window of my faculty host.When I arrivedin Santa Cruz at the end of my trip, I parked my rental carin the parking lot that the map showed was nearest to thesite of my interview. When I got out of the car, I foundmyself surrounded by a forest of redwood trees. There wasno building in sight. I had no idea where to go. At thatmoment, I realized that I had an easy decision: if the Uni-versity of California, Santa Cruz (UCSC), made me anoffer, I would accept on the spot, no matter what it was.UCSC was an “experimental” campus and was in the

third year of its existence. The theoretical chemist TerrellHill, who recruited me, likened it to “an embryonic Cam-bridge or Oxford,” with its residential college system. Acompeting view was that it was an embryonic Haverfordor Reed, focusing on undergraduate liberal-arts education.Fortunately, I was idealistic, fearless, and naïve. As anassistant professor, I taught five courses and served on




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eight faculty committees (and chaired five of them). I cen-tered my life around the lab (Fig. 1), and when the timerrang, I went to class.When class was over, I headed back tothe lab, where I asked students to wait for a break in myexperiments to ask questions about my lectures. The ivo-ry-tower atmosphere and relative isolation of UCSC werein a way liberating; there were not a lot of “experts” aroundto discourage you from going in unusual directions.My view of the ribosome in 1968 was a fairly conven-

tional one for the time: the ribosome was believed to besort of a multienzyme complex with lots of proteins, eachof which contributed to a specific ribosome function. Idecided to pick a couple of interesting proteins out of thefifty or so that had been identified and, like a good enzy-mologist, work on their structures and functions. The firstquestion was which proteins to work on. Having a back-ground in protein chemistry, I decided to bombard theribosome with a wide range of chemical modificationreagents known to react with the active sites of enzymesand test the ribosomes for loss of specific activities, such astRNA binding or peptidyl transferase activity. When Ifound a loss of activity, I would then useMasayasu Nomu-ra’s in vitro reconstitution approach (1) to identify whichprotein had been functionally modified by the rescuingactivity of the individual purified proteins. We tried all ofthe usualmodification reagents and then some. One of themorememorable was the histidine-specific reagent diazo-nium-1H-tetrazole, which had to be synthesized on thespot because of its instability. I carried out a small-scalesynthesis in a large test tube that was too tall for the lengthof my micropipette. In a display of youthful hubris to fixthe problem, I etched a line halfway up the tube with mydiamondpencil, touched amolten glass rod to the line, andstruck the top of the tube sharply with the other end of the

glass rod. I should have anticipated the potential chemicalproperties of a heterocyclic aromatic ring compound con-taining six nitrogen atoms and only a single carbon atomin its structure. Fortunately, the shards of glass unleashedby the explosion somehowmissedmy unshielded eyes. Forthe next weeks, anyone walking through that part of thelab could hear sharp popping noises coming from the flooras they stepped on the thousands of tiny crystals ofdiazonium-1H-tetrazole.Strangely, the initial result of screening dozens of mod-

ification reagents was negative. None of the reagentsknocked out ribosome activity, until my graduate studentGeorge Thomas tried photooxidation induced by the dyeRose Bengal, which had been used successfully to targetactive-site histidines in some enzymes. Rose Bengal finallycaused inactivation of protein synthesis, including bindingof tRNA to ribosomes. George’s reconstitution experi-ments showed that modification of some ribosomal pro-teins was partly responsible for the loss of activity, but alsothat there had been functional modification of 16S rRNA.We were puzzled and troubled by this result until wefound reports in the literature that guanine could also betargeted by Rose Bengal photooxidation. Around thistime, in the spring of 1972, an undergraduate by the nameof Brad Chaires came to my office to confess that he hadmade little headway on his senior thesis in my lab andneeded to finish a project to graduate within the followingcouple of months. I suggested that he try to intentionallytarget the ribosomal RNA using kethoxal, a guanine-spe-cific reagent. The following week, he came back to myoffice with an amazing result: kethoxal had completelyinactivated the ribosome!Moreover, reconstitution exper-iments confirmed that the inactivation was indeed causedby modification of the ribosomal RNA; the proteins fromthe kethoxal-modified ribosomewere 100% active. He alsoshowed that binding tRNA to the ribosomes prior tomod-ification protected them from inactivation and that inac-tivation was caused by modification of only a handful ofguanines out of the more than 400 present in 16S rRNA.This was our first clear result, but not at all what wewanted to hear. No one would believe that RNA wasinvolved in enzymatic function, much less in the functionof such a complex and fundamental structure as the ribo-some. Furthermore, the ribosomal RNAs were 2 orders ofmagnitude larger than any molecule that had been char-acterized by enzymologists.Worse, I knewnext to nothingabout RNA chemistry.We next wondered which nucleotides were the ones

whose kethoxal modification was causing loss of ribosomefunction. Unfortunately, the nucleotide sequence of 16S

FIGURE 1. The author in his laboratory at UCSC as an assistant pro-fessor in 1970.




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rRNAwas incomplete, and therewas no easyway to deter-minewhich of itsmore than 1500 basesmight be function-ally important. Our direction came from the observationthat tRNA protected the ribosomes from inactivation bykethoxal: the critical guanines must be among the tRNA-protected bases. The first challenge was to figure out a wayto identify the guanines that were being modified bykethoxal and then to identify the ones that were protectedfrom modification by tRNA. We succeeded with the firstpart of the plan within a year or so, but the second parttookmore than an additional decade. The initial approachemerged during a conversation with Jim Dahlberg, whowas visiting fromWisconsin. Jim had also been a postdoc-toral fellow at the MRC, where Brian Hartley had intro-duced “diagonal thinking.” Whatever problem you werestudying, there always seemed to be a two-dimensionalpaper electrophoresis “diagonal” solution. Jim asked me ifthe kethoxal reactionwas reversible, and I immediately gotit. If we cut the kethoxal-modified 16S rRNA with RNaseT1, which cuts specifically at G, the modified guanineswould be resistant to the nuclease. The result would be amixture of all of the oligonucleotides ending in G, exceptfor the kethoxal-modified sites, which would give a pair oftwo adjacent oligonucleotides connected by an internalkethoxal-modified G.The first step of the diagonal method consisted of run-

ning the T1 digest of uniformly 32P-labeled RNA by paperelectrophoresis on DEAE paper in the first dimension,separating the oligonucleotides according to their size andbase composition. I then cut out the strip containing thefirst dimension and removed the kethoxal from the mod-ified Gs by treatment with mild base while the oligonu-cleotides retained their positions bound to the DEAEpaper. Next, I soaked the paper with RNase T1, which cutbetween the pairs of oligonucleotides at the sites that hadbeen modified. The strip was then sewn onto a fresh sheetof DEAE paper at right angles to the direction of the firstdimension, and the electrophoresis was rerun. When Iturned on the darkroom right after developing the firstautoradiogram, I was ecstatic. It showed a very dark diag-onal line from all of the oligonucleotides that were unaf-fected by the redigestion step, but pairs of off-diagonalspots appeared, corresponding to the oligonucleotidessurrounding the sites of kethoxal-modified guanines. Irealized that we now had the means to identify the sites ofkethoxal modification, which we could soon put into thecontext of the emerging complete sequence of 16S rRNA.I then fell into a months-long slump, during which I

could not reproduce my initial success, and my euphoriafaded into depression and self-doubt. The problem was a

dark streak running up from the origin of the seconddimension, which had been absent in my first experiment.I began repeating the experiment, testing every possibilityI could think of. This required growing cells in millicuriesof [32P]orthophosphate, isolating blazingly hot ribosomesand ribosomal subunits and carrying out the two-dimen-sional diagonal electrophoresis procedure. Each trial tookabout a week and a half, and each time, the dreaded streakreappeared, even though I had been carefully using all ofthe same solutions that I had used in the successful trial. Indesperation, I began remaking all of my reagents and buf-fers. When I made up a fresh bottle of water-saturatedphenol that I was using to extract the RNA from the ribo-somes, the streak vanished! Later that year, in the breakfastline at a meeting at Squaw Valley, I found myself standingnext to Fred Sanger, whom I had not seen since my post-doctoral days at the MRC. He asked what I was up to andlistened with rapt attention as I described my diagonalmethod, asking about experimental details such as pH,temperature, electrophoresis conditions, and so on.WhenI was finished, he smiled and said, “I’ll bet you tried a lot ofthings that didn’t work!” His remark, although at firstseemingly telepathic to me, was a revealing look into thegenius of one of the greatest experimental scientists of thetwentieth century. I have since then repeated his wordscountless times to students and postdoctoral fellows and,of course, to myself during those moments of quiet des-peration that we repeatedly face in our laboratories.

The ribosome teaches in silence.—Carl Woese

I began sequencing the off-diagonal oligonucleotides,which led to the identification of the sites of kethoxalmod-ification of 16S rRNA. This was helped enormously by amethods chapter (2) written by Bart Barrell, who had beenSanger’s technician during the development of RNAsequencing, and also by desperate phone calls to HowardGoodman, who had been a fellow postdoc scientist inGeneva and had taken a faculty position at University ofCalifornia, San Francisco (UCSF). During this time, I wasplaying with the Randy Masters Group. I would start anelectrophoresis run before dashing off to rehearse Randy’scomplex polyrhythmic arrangements and then return tothe lab late at night to pull the racks out of the electropho-resis tanks. Some highlights were opening for the DukeEllington Orchestra at the Del Mar Theater in Santa Cruzin 1974 and a concert at the Santa Cruz Civic Auditoriumalong with Bobby Hutcherson and Eddie Henderson andtheir bands. As my oligonucleotide sequences took shape,I began to doubt my skills as an RNA biochemist. Many ofmy sequences were in disagreement with the published




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ones. I was consoled by a phone conversation with CarlWoese at the University of Illinois at Urbana-Champaign;he had begun sequencing the RNase T1 oligonucleotidesfrom different bacterial species to establish their phyloge-netic relationships. This project led him to the discovery ofthe Archaea, the third domain of life. When I mentionedmy discrepancies, Carl confirmed that my sequences werecorrect. Furthermore, he had found that over half of thepublished sequences of oligonucleotides of length greaterthan five were incorrect. His voice strained with barelycontained anger. “These sequences are sacred scrolls.They should be entrusted only to those who appreciatewhat they mean,” he said. I was stunned. Everyone in thefield used the partially finished 16S rRNA sequence as animportant resource and assumed that it was correct andwould soon be complete. Work had begun on attemptingto fold it into a secondary structure, amajor step toward anunderstanding of the ribosome structure. All of this wasnow cast in doubt. I gradually began to come to termswiththe possibility that we might have to determine thesequence ourselves, which would entail a staggeringamount of very challenging work.An unexpected and exciting surprise from my conver-

sation with Carl was that my oligonucleotide sequenceswere unusually rich in universally conserved sequences. Atthe time, conventional wisdom dictated that the mostimportant sequences in ribosomal RNA would be thebinding sites for ribosomal proteins (the putative “func-tional” molecules of the ribosome), yet the conservedkethoxal-reactive sites were exposed to the solvent, aswould be expected if the ribosomal RNA itself were func-tionally important. Therefore, I was thrilled when I wasinvited to contribute a chapter to the Cold Spring Harborvolume Ribosomes (edited by Masayasu Nomura, AlfredTissieres, and Peter Lengyel) based on a meeting where Ihad presented my kethoxal sequence results for the firsttime. However, as I was nearing completion ofmy chapter, Ireceived another letter fromNomura, uninvitingme to con-tribute my chapter because of space limitations. This wassurely the last andonly time inmycareer that Iwouldactuallybe disappointed not to have to write a review article.Association of the two ribosomal subunits was also

inactivated by the reaction of kethoxal with conservedsequences in 16S rRNA, as discovered by two undergrad-uates, Winship Herr and Nora Chapman.We also discov-ered that peptidyl transferase, the peptide bond-formingcatalytic activity of the ribosome, was abolished bykethoxal modification of 50S ribosomal subunits. Theseexperiments, carried out by another undergraduate, RobAtchison, were never published because we had difficulty

separating the 23S rRNA from the 50S proteins of thekethoxal-modified 50S subunits. Later, it seemed likelythat this difficulty was caused by polymerization of ourstock sample of kethoxal during storage. However, thisresult bolstered our convictions that ribosomal RNA wasindeed functional, as did the finding by yet another under-graduate, Jim Breitmeyer, who found that a reactive electro-phile attached to the �-amino group of an aminoacyl-tRNAbecame attached to 23S rRNA, not to a ribosomal protein.In the summer of 1975, coming up for my first sabbati-

cal, I received a phone call from Christine Guthrie, whohad just returned from the Gordon Research Conferenceon Nucleic Acids. She said, “Allan Maxam from WallyGilbert’s group and Bart Barrell from Fred Sanger’s groupannounced that they are sequencing DNA on gels! Maybeyou could use this approach to sequence ribosomal RNA.”Larry Soll at the University of Colorado in Boulder wasknown to have isolated a � transducing phage that hadpicked up some ribosomal RNA genes from Escherichiacoli, so there was a possible source of the DNA. I wrote toFred Sanger, asking if he thought the new sequencingmethod might be applicable to doing the whole ribosomalRNA and if I might be able to visit his lab to learn it. Hisreply was encouraging (Fig. 2). As an aside, it is interestingto note that the state of the art of DNA sequencing inSeptember 1975 was such that “ . . . one should be able todeduce a sequence of about 50 residues every few days . . . ”I spent the first fewmonths of my sabbatical in Berlin at

theMax Planck Institute forMolecular Genetics inHeinz-GunterWittmann’s department, collaborating with RogerGarrett to apply the kethoxal diagonalmethod to themap-ping of the binding sites for proteins L5, L18, and L25 on5S rRNA. As one of the few people who worked in the labafter 5 p.m., one evening, I heard the unmistakable voice ofWolfman Jack as I wandered the halls. I traced its source toan Armed Forces Network program coming from a bigGerman table radio sitting in a tiny closet of an office occu-pied by Jurgen Brosius, a graduate student who wassequencing ribosomal proteins under Brigitte Wittmann-Liebold forhis doctoral research. Soon,wewere goingout forlate-night dinners and beers, as Jurgen introducedme to theHundekehle, the Zwiebelfisch, and other after-hours venuesof the fascinating enclosed city ofWest Berlin.Moreover, the experiments that Roger and I were doing

were actually working, so Roger advised me to let the peo-ple in the business office know that I would be prolongingmy stay for another month. However, Wittmann himself,a devoted protein chemist, was not thrilled by ribosomalRNA. One afternoon, he stormed into the lab while I wasin the middle of an experiment, irately accusing me of




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manipulating his business office into giving me moremoney. I explained that I was happy to support myselffrom my own grant from the NIH, but that Roger hadinsisted that I simply inform the business office that Iwould be staying another month. Wittmann then asked,“And you really believe that ribosomal RNA is importantfor function? Even peptidyl transferase?” By now, he wasbecoming quite red in the face and stormed out of thelaboratory.My thoughts began to turn toGeneva, the nextstop on my sabbatical, where Alfred Tissieres had offeredme bench space for a few months.I had no idea a priori what I was going to do in Alfred’s

lab, especially because he no longer worked on ribosomes.This was resolved during my first day in the laboratory. I

was describing to the postdoctoral fellow Jack Greenblattthat I was thinking about sequencing ribosomal RNAusingDNA sequencing, and he said, “Oh, Joel Kirschbaumhas a � transducing phage with ribosomal RNA genes onit.” When I asked where Joel Kirschbaum worked, Jackreplied, “He’s right across the hall.” I was stunned. Next,Joel was handingme a sample of �drifd18, which containedthe entire rrnB operon, and explaining how to grow it andextract its DNA. Soon it was time to head to Cambridge tolearn DNA sequencing. Fred Sanger had arranged for meto work alongside his right-hand man, Bart Barrell, whowas now part of the group sequencing the completegenome of the phage �X174. Fred had reserved rooms forme in King’s College. At the end of my first day in Fred’slab, I returned to my rooms to find an elderly gentlemanfast asleep in my bed. Fred was greatly amused by thisnews, proudly reporting to everyone in the lab the “extras”that they had laid on for my stay at King’s. Working withBart was intense fun. At that time, micropipetting wasdone with Microcaps, which were small calibrated capil-laries that you attached to a rubber adaptor and a longpiece of rubber tubing for mouth pipetting. After additionof each 32P-labeled sample, Bart flicked the usedMicrocapinto awastebasket sitting in themiddle of the bay about sixfeet away. The majority of the Microcaps hit their target;the rest formed a blazingly radioactive halo circling thebase of the wastebasket. Bart was a superb teacher, and ina few days, I was admiring my first successful sequencingautoradiogram (Fig. 3).Among the postdoc fellows working on the �X174 pro-

ject in Fred’s lab wasWayne Barnes, who askedme if I had“clones” of the ribosomal RNA genes. When I mentionedJoel Kirschbaum’s �drifd18, he was astonished at my clue-lessness. “No, Imean a clone!Where’s the phageDNA?” heasked. I phoned Pia Malnoe, Alfred’s Swedish technicianin Geneva, and asked her to find a beaker on the secondshelf in the cold room and to send the tube of DNA to mein Cambridge via air mail (FedEx had yet to come intoexistence). When the �drifd18 DNA arrived, Wayne hadme cutting it with restriction enzymes and cloning thepieces into the ColE1 plasmid (the naturally occurringancestor of pBR322 and many other modern cloning vec-tors). Within a couple of days, I was making stabs of theclones and putting them into a package that I addressed tomyself in Santa Cruz.

Everything you know is wrong.—The Firesign Theater

From the phone calls fromCarlWoese and Chris Guth-rie, Fred Sanger’s letter, Joel Kirschbaum’s phage, andWayne Barnes’s cloning expertise, I had the eerie sensa-

FIGURE 2. Letter from Fred Sanger with encouraging words forsequencing the ribosomal RNA genes by DNA sequencing.




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tion that everything was falling into place guided by amys-terious force. The final step was a crucial conversationwith Jurgen Brosius at a sidewalk cafe in Geneva, when Iexplained to himwhere thingswere headed and persuadedhim to come do postdoctoral research with me in SantaCruz. Jurgen received a Fogarty Fellowship from the NIHfor this, and he arrived in the lab the following year. Wehad only a tiny lab of about 600 square feet, but we man-aged to convince the department to give us an additionalsmall room where we could set up DNA sequencing gelrigs. We had two gel boxes made and ran them off asingle power supply, but Jurgen soon complained thatwe needed two more and then four more, until we ranout of supply money. Carl Woese stepped in and gener-ously had eight more built for us at the University ofIllinois, finally giving us a total of sixteen gel boxes.Jurgen was a force of nature, working day and night withan undergraduate, Tom Dull. They came to the lab inthe morning, loaded sixteen warm pre-run sequencinggels, reloaded them a few hours later for short reads (toread the end-proximal sequences), and then stoppedthem and put on the x-ray film in the early afternoon.They then mounted fresh gels and pre-ran them whilethey ate and slept, coming in late afternoon to beginloading the next round of samples, finishing after mid-night. Sequencing technology was far from perfect atthat time. We had settled on the Maxam-Gilbert chem-

ical sequencing method, a challenging technique thatoften failed to distinguish between Cs and Ts. Therewas readable sequence in about one out of four gel runs.However, with more experience, we started to be able toread 200 or more nucleotides at a time. We even had tomake our own [�-32P]ATP for end labeling DNA frag-ments and isolate our own restriction enzymes. Ourfirst long sequence reads showed that the publishedpartial sequence of 16S rRNA had mistakes not only inthe sequences of the RNase T1 oligonucleotides, butalso in their ordering within the sequence. In all, itcontained more than 200 errors. We presented thesequence at the ribosome meeting in Salamanca in June1978, hosted by the great Spanish antibiotic guru DavidVazquez. Late at night after much partying, Vazquez wasin fine form, on the loose in the bar with a dangerous-looking saber, demonstrating some kind of African sworddance. Awellmeaning attendee talked him out of the knifeby trading in it for a pair of scissors. Vazquez then begancutting chunks of hair off the heads of scientists in the bar,leaving a stream of bald patches in his wake, urged on by acheering crowd.When he spottedme (Fig. 3), a huge cheerwent up as he cut off a two-foot-long fistful of hair, leavingme lopsided but otherwise mostly intact.By the time we finished the sequence of E. coli 16S

rRNA, we were, of course, well into the sequence of 23SrRNA and all of the rest of the DNA sequence of the rrnBoperon. However, Carl Woese’s paper on 5S rRNA hadconvinced me that the only way to figure out the second-ary structures of the large ribosomal RNAs was tosequence several phylogenetically distinct versions of 16Sand 23S rRNAs. Thus, in parallel with finishing the E. colisequences, JoAnn Kop, Ramesh Gupta, Alexei Kopylov,Ginny Wheaton, and others in my group begansequencing the large ribosomal RNAs from Bacillus,Haloferax, and other species. In the meantime, otherlaboratories were beginning to sequence ribosomalRNA genes from many sources. Before long, it becamedifficult just to keep them straight because no computersoftware had been written to deal with alignment ofmultiple sequences. In fact, computer technology wasbarely able to deal with storage and retrieval of singlesequences. Robin Gutell was entering sequences on amassive VAX750 (a “minicomputer” the size of a smallrefrigerator) using a remote terminal until we pur-chased our first lab computer, a Sun Microsystemsworkstation with 2-MB main memory ($15,000) and an86-MB hard drive (another $15,000). When I informeda computer-savvy colleague that we had just bought an86-MB hard drive, he laughed, “You’ll never use all of

FIGURE 3. The author on sabbatical in Cambridge in April, 1976,holding the autoradiogram of his first successful DNA sequencinggel.




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that storage space!” The alignment problem was solvedby a computer sciences undergrad, Ted Goldstein, whodid his senior thesis project in our laboratory. Afterasking Robin a lot of questions, Ted disappeared for acouple of weeks and returned with a program he calledSTREAM, which was, quite possibly, the very first mul-tiple-sequence alignment editor. Ted had ripped apartthe source code for the UNIX operating system andtweaked the vi text editor to create his program, whichwe used successfully for many years.Using STREAM and fistfuls of colored pens, we began

dissecting the secondary structures of the large ribo-somal RNAs in collaboration with Carl Woese, who hada wealth of phylogenetically diverse T1 oligonucleotidesequences to add to the rapidly developing database ofcomplete sequences. We looked for compensatory basechanges, i.e. complementary sequences in which muta-tional changes occurred in both strands such that Wat-son-Crick pairing wasmaintained. By the time we beganon the secondary structure of 23S rRNA (2904 nucleo-tides), we waited until we had three complete sequencesand then used a simple visual algorithm that we called“red dot-green dot.” Wherever there was a transversion,we put a red dot; transitions got a green dot. We thenlooked for complementary sequences that showed mir-ror-symmetric patterns of red and green dots. Thisapproach remains the simplest and most powerfulmethod for determining secondary structures of RNA,short of x-ray crystallography.The next challenge was to develop a better tool to

study ribosomal RNA (faster and more comprehensive)to be able to study the interactions of all of the bases(and possibly the riboses and phosphates) with thefunctional ligands and proteins of the ribosome. Afterhearing a talk in which primer extension was being usedto identify breaks in DNA, I realized that we might beable to use this approach to map sites of modification bykethoxal and other reagents in the ribosomal RNAusing reverse transcriptase. A similar method was beingdeveloped in parallel in Tom Cech’s lab at the Univer-sity of Colorado in Boulder unbeknownst to us. Aftergetting advice on the use of reverse transcriptase fromNorm Pace at Indiana University, I tried it on some 16SrRNA that I had modified with several reagents andbingo! There were the bands at the modification sites.This caught the attention of three graduate students,Danesh Moazed, Seth Stern, and Ted Powers, whomdivine providence had sent to our lab for this purpose.After optimizing the method, Danesh set out to mapfunctional ligands, and Seth and Ted began to map the

binding sites for the ribosomal proteins. A few dayslater, I was working at my bench when I was confrontedby the three of them. “What are you doing?” they asked.Taken aback, I started to explain that I was makingsome ribosomal protein stock solutions. “No. We aredoing that,” they said. “You are writing the papers.” Istarted to argue but stopped when I realized I would notwin that argument. These three students exploded withexciting results for the next several years. They mappedthe binding sites for the tRNAs, protein synthesis fac-tors, ribosomal proteins, and antibiotics. Out of thiscame the hybrid-states mechanism for translocation,the placement of antibiotics in functional sites in theribosomal RNA, and an initial model for the three-di-mensional folding of 16S rRNA. The universal conser-vation of the ribosomal RNA sequences protected bythe tRNAs and factors provided overwhelming evidenceto convince us that ribosomal RNA was at the heart ofribosome function.However, Wittmann’s protein-centric point of view

continued to represent thinking in the field at that time.Indeed, it was representative of the convictions of bio-chemists in general. After giving a talk on the functionalrole of ribosomal RNA at Berkeley, a colleague confided tome that a distinguished biochemist had remarked, “Whata crackpot idea!” I was reminded of the Firesign Theater’slandmark album Everything You Know Is Wrong, ananthem for the state of the ribosome field. Of course, therewas an upside to this: we had very little competition study-ing the functional role of ribosomal RNA for about a dec-ade. However, once self-splicing introns were discoveredby Tom Cech, Norm Pace, and Yale University’s SidneyAltman in the early 1980s, it quickly became respectable towork on ribosomal RNA function. The final strawoccurred after my talk at the 1987 Cold Spring HarborSymposium, when Phil Sharp, who is at theMassachusettsInstitute of Technology (MIT), said tome, “So, Harry, whydon’t you nail it?” I stared at him blankly. “What do youmean, Phil?” I was so immersed in my own belief in thefunctional role of ribosomal RNA that I was unable tosee the question from the outside. There was so muchcircumstantial evidence that I had long since stoppedquestioning it.I then remembered an experiment that had been

done by a postdoctoral fellow, Ludwika Zagorska (laterZimniac). As a control in a peptidyl transferase assayshe was doing, she had included the strong ionic deter-gent SDS in one of her reactions. In place of the fMet-puromycin product, a smear appeared in that lane. Itoccurred to me that the smear might have actually been




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the product, whose migration on paper electrophoresiswas distorted by the presence of the detergent. When Igot back to the lab from Cold Spring Harbor, I treatedsome ribosomes with SDS but then removed the SDS byethanol precipitation from the extracted ribosomesbefore carrying out the reaction. As I had guessed, abeautiful product spot appeared: the SDS-treated ribo-somes were fully active in peptide bond formation!Next, I upped the ante by adding some proteinase K, astandard method for deproteinizing nucleoproteinsamples. Again, there was full activity. Finally, I addedan equal volume of phenol to the SDS/proteinaseK-treated ribosomes and vortexed them continuouslyfor half an hour. The E. coli ribosomes finally lost activ-ity with this treatment, but the Thermus aquaticus ribo-somes retained their full activity. Although not all of theribosomal protein was removed by this treatment, Idecided that it was interesting enough to publish. I hadintended to submit it as a letter to the Journal of Molec-ular Biology, but Christine Guthrie, who was on sabbat-ical at UCSC fromUCSF, was so excited about the resultthat she convinced me to send it to Science. I was sur-prised at the RNA community’s reaction to this result,but it was a relief not to have to justify my passion forribosomal RNA after that (Fig. 4).

Just get the spots.—Brian Matthews

At each stage, our desire to know the three-dimensionalstructure of the ribosome grew more intense. Modelingexercises in our lab and inRichard Brimacombe’s lab at theMaxPlanck Institute forMolecularGenetics in Berlin gaveus a general sense of where the interesting things were inthree dimensions (in fact, we fairly accurately predicted

the arrangement of the three tRNAs in the ribosome).However, these attempts always came up short of what wereally wanted: a crystal structure of the whole ribosome,with its mRNA and tRNAs in place. Alex Rich had anotorious policy of requiring his students at MIT tospend their first year attempting to crystallize ribo-somes. I began thinking that everyone in the fieldshould spend, say, 5% of their effort on ribosome crys-tallization. In the late 1980s, Ted Powers and I began aneffort to crystallize T. aquaticus 70S ribosomes withoutmuch success until one day when Ted found a beautifulcrystal in one of his drops. I phoned Steve Harrison,who had solved the structures of viruses in his lab atHarvard, and he flew to Santa Cruz to retrieve the crys-tal for analysis. A few days later, Steve phoned with thesad news that it was a salt crystal, most likely magne-sium ammonium phosphate. I was embarrassed butpuzzled. I had carefully avoided having any phosphatepresent in our drops for this very reason. It turned outthat the polyethylene glycol we were using as precipi-tant was contaminated with phosphate.A few years later, I heard fromMarat Yusupov, whowas

then working in Strasbourg at the CNRS lab. He hadnoticed our paper on in vitro reconstitution of the head ofthe 30S subunit, done by Ray Samaha, and suggested com-ing to Santa Cruz to crystallize it for structure determina-tion. I recognizedMarat’s name from the paper from Rus-sia in which crystallization of the Thermus thermophilus70S ribosome had been reported (3). I replied, “Why don’twe try to solve the whole thing?” A few months later,Marat and hiswife, Gulnara, arrived in SantaCruz to beginthe project. Unfortunately, the crystal form that had beenobtained in Russia diffracted to no better than about 20 Å.However, after a few months in Santa Cruz, the Yusupovsfound a new crystal form of the T. thermophilus 70S ribo-some in the I422 space group, and before long, it was dif-fracting to around 12 Å. On a seminar visit to Eugene, Itold Brian Matthews about what we were up to andexpressed to him my worries about the next steps. “Brian,if we get these crystals to diffract well, how on earthwill webe able to solve the structure? The ribosome is much toobig to use heavy atoms for phasing.” Brian’s reply was reas-suring. “Just get the spots,” he said. “You’ll figure out howto phase them.”Around this time, I met Jamie Cate, a young crystallog-

rapher who had just solved the structure of the P4–P6domain of the group I intron as a graduate student in Jen-nifer Doudna’s lab at Yale. When I asked him whether hemight be interested in solving the ribosome, Jamieanswered me with raised eyebrows. He joined our small

FIGURE 4. The author in his laboratory in 1991 doing the peptidyltransferase experiments.




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group, which we calledMission:Impossible.We knew thatwe were up against some of the best crystallographers inthe world, but among ourselves, we had an almost per-fectly complementary set of researchers. We spent manylong nights at the Advanced Light Source, running on cof-fee, donuts, and barbecued ribs from the legendary Everett& Jones Barbecue inWest Berkeley. The Yusupovs contin-ued to improve the diffraction properties of the crystals,and Jamie began to work on phasing the ribosome at lowresolution. He started with the molecular envelope of theE. coli 70S ribosome from Joachim Frank’s cryo-EMreconstruction, packing it with Gaussian spheres thatwere matched to the average scattering power of the ribo-some, essentially filling the ribosomal envelope with BBs.One day, he discovered a molecular replacement solutionthat provided phases to about 20 Å resolution, allowinghim to calculate an electron density map that looked like aribosome. The proof that we had independent phasescame from a difference map from ribosomes that had atRNA bound to the A site versus ones with a vacant A site.We crowded around the SGImonitor to look at the result.A chubby L-shaped density appeared in exactly the posi-tion that we had predicted for the A site from our bio-chemical work! Thiswas the ahamoment.With these low-resolution phases, Jamie could then calculate higherresolution phases using the heavy-atom cluster tantalumbromide. From those phases, he could find individual irid-ium hexammine heavy atoms for phasing to the limits ofour diffraction data, which the Yusupovs improved first to7.8 Å and then to 5.5 Å. Meanwhile, the Steitz andRamakrishnan groups at Yale and Cambridge, respec-tively, and the Yonath group in Israel were solving thestructures of the ribosomal subunits at all-atom resolu-tion. Although at lower resolution, we could see the wholething: how the subunits fitted together with their dozenintersubunit bridges; how the tRNAs bound to the A, P,and E sites of the ribosome; and the path of the mRNAthrough the ribosome. As we anticipated, all of the func-tional sites were made almost exclusively of ribosomalRNA; the ribosomal proteins were scattered mainlyaround the periphery of the ribosome.However, just as the

structure was unfolding, Jamie was recruited to a facultyposition at the Whitehead Institute for Medical Researchand the Yusupovs to positions in Strasbourg. Eventually,we rebuilt our crystallography group at Santa Cruz withthe arrival ofAndrei Korostelev, Sergei Trakhanov,MartinLaurberg, Jie Zhou, and others, and we began solving all-atom structures of the 70S ribosome trapped in differentfunctional states.In parallel, we began to complement the static snap-

shots of the crystal structures of the ribosome withstudies of its structural dynamics in solution usingFRET methods, including single-molecule FRET, pio-neered by the postdoctoral fellows Robyn Hickersonand Dmitri Ermolenko, in collaborations with BobClegg and Taekjip Ha at the University of Illinois andtheir co-workers. A collaboration with Nacho Tinocoand Carlos Bustamante at the University of California,Berkeley, has provided yet another view of the behaviorof single ribosomes using optical tweezers. Paradoxi-cally, as methods have become more and more power-ful, with higher and higher resolution, the problem ofunderstanding the molecular mechanism of action ofthe ribosome has become increasingly challenging andever more complex. This has proved to be a happy sit-uation, making it possible to spend my entire career ona single problem. Fortunately, the pursuit of this prob-lem continues to be a rewarding one, illuminating oneof the deepest and most central mechanisms in all ofbiology. Or, as the headline writers for the OaklandTribune would have put it, “Scientists Continue Searchfor Secret of Life.”

Address correspondence to: [email protected].

REFERENCES1. Nomura, M., Mizushima, S., Ozaki, M., Traub, P., and Lowry, C. V. (1969)

Structure and function of ribosomes and their molecular components. ColdSpring Harbor Symp. Quant. Biol. 34, 49–61

2. Barrell, B. G. (1971) in Procedures in Nucleic Acids Research (Cantoni, G. L.,and Davies, D. R., eds) Vol. 2, pp. 751–779, Harper & Row, New York

3. Trakhanov, S., Yusupov, M., Shirokov, V., Garber, M., Mitschler, A., Ruff, M.,Thierry, J. C., and Moras, D. (1989) Preliminary X-ray investigation of 70Sribosome crystals from Thermus thermophiles. J. Mol. Biol. 209, 327–328




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Harry F. NollerBy Ribosome Possessed

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