Introduction

THIS BOOK WAS WRITTEN FOR SCIENTISTS of all kinds. It is unapologeticallyhistorical. But, you say, the world of biology is rocketing ahead at a pace

undreamed of even a decade ago. The advancing technological age in biologythat began roughly 35 years ago with the “recombinant DNA revolution” nowpresents a daily mountain of new information. So why be so misguided in themidst of this whirlwind of the new as to turn out a history? And why a historyof RNA?

Consider this: How in the next couple of decades are newcomers to biol-ogy going to learn, and how and what are established scientists going to teachthem? Already, virtually all college-age students have had exposure, oftensince grade school, to the mantra “DNA makes RNA makes protein.” In thiscomputer age, the notion that biology is an information science and thatDNA is the library seems a congenial concept to most who are inclined towardan analytical/scientific career. Perhaps the sensible and necessary course toproperly prepare declared biology students and analytically trained “transfers”(mathematicians, physicists, engineers) is first to serve up a predigested cate-chism of settled conclusions achieved in the 20th century by “wet” laboratoryexperiments. With this concise biological “periodic table” under command,the newcomer then can be efficiently prepared to deal with the rapidlyadvancing technology both for doing experiments and for collecting and ana-lyzing to a useful purpose the enormous quantity of data that emerges fromtoday’s genomic, proteomic, and computationally enhanced microscopicinvestigations.

It is by no means my intent to deflect teachers/scientists (mostly young,under 40 years of age) who must carry out the indispensable task of gettingstudents ready to enter today’s biology world. Rather, my aim in writing this

1

book is to provide a supplement in historical form—both to the younger gen-eration of scientists and teachers and through them to incoming students—that describes how we first learned some of the molecular fundamentals ofbiology in the days of the “hands-on wet laboratory.”

One can legitimately argue whether a 2011 biology student “needs” toknow any pre-1990 history. I am not prepared to defend vigorously theaffirmative in this debate. But I will argue that many may choose to knowhow we came to know all that we did in the era before commercial kitsand genomic sequencing took hold. Many of today’s major questions (e.g.,about how messenger RNAs [mRNAs] are formed and about how gene con-trol is exercised in eukaryotes) are the same questions that were pursued in1962–1980. More detailed answers to these questions are arriving today atbreathtaking speed, but fundamentally informative and important answerscame between 1962 and the early 1980s. How these still-central questions firstarose, and how early experiments were structured and answers obtained, itseems to me, ought to be at least available in usable form for teachers and,most of all, the curious students of today.

I have been privileged to listen in on a number of “after hours” (read“faculty club cocktail hour”) discussions among physicists. Both elders andyoungsters in that community seem able to discuss where ideas, questions,and answers came from, easily back to Maxwell and his equations. Biologicalscience, specifically the role of RNA in current and past life on this planet,also has a history worth knowing, I believe.

This history begins with the following questions: How did macro-molecules finally become recognized as the necessary starting place for firstlearning about biology and now teaching biology? And why was RNA the late-comer in this overall picture?

The bold discovery by James Watson and Francis Crick of the structure ofDNA, often told, and well told, by the protagonists themselves, is frequentlyrecited as the “start” of molecular biology. And if one watershed discovery isto be chosen as the “beginning,” that discovery is it. But there was a precedinghalf-century struggle of genetics and physical biochemistry that prepared atfirst a small group of scientists to grasp what the Watson-Crick structureboth predicted and demanded but did not answer. For the discovery of theDNA structure and following discoveries to occur, biology/biochemistryhad to take on macromolecules. The reign of organic chemistry (and recalci-trant organic chemists) as the main route to understanding life had to be atleast momentarily sidestepped so that large molecules, poorly understoodand comparatively difficult to study, could become the major research focus.How molecular biology involving macromolecules emerged from these early20th-century battles is fascinating history.

2 Introduction

The double helix discovery instantly revealed, through what Crick in hisbook What Mad Pursuit called “such a beautiful structure” (p. 60), how themolecule worked in inheritance. But the Watson-Crick revelation also lit thefuse that led to uncovering the centrality of RNA to life. The miracle years of1955–1961—just 50 years ago—finally saw RNA recognized to be not mono-lithic but a collection of different types of molecules with specific functions.Courtesy of the insight of Francois Jacob and Jacques Monod, biological spe-cificity among different cells, formally a completely opaque problem, couldnow at least provisionally be explained by controlling the synthesis of specificmRNAs.

The establishment of RNA function—first by discovering how geneticinformation is transferred into a readable form and then by proving the inti-mate roles of RNA in translation—led shortly to deciphering the universalgenetic code, the first breakthrough toward which Marshall Nirenberg carriedout in 1961. But all of these heady achievements were accomplished (largely)with bacteria and their viruses.

As biologists took these ideas to eukaryotic cells, first with culturedhuman cells, RNA remained the major focus. Throughout the 1960s and1970s, a new world of macromolecular genetics was unearthed throughstudies of eukaryotic RNA. Unknown at the time, storage of information inthe DNA of eukaryotes was very different from that of bacteria. Simplycopying DNA into RNA did not suffice for genetic function. Primary RNAtranscripts required molecular carpentry of various kinds—generically termedRNA processing—to produce functional RNAs. This era culminated in 1977with the discovery of pre-mRNA splicing to produce functional mRNA.Both the complicated machinery for digging out the primary transcript aswell as the processing to make a specific mRNA opened our eyes to additionalpoints at which regulation of mRNA might occur. All of this was well estab-lished before facile genomic sequencing confirmed these conclusions.

Soon thereafter (1979–1981), a second bombshell burst. Chemical catal-ysis can be performed by pure RNAs, most often held in the proper tertiarystructure inside cells by protein scaffolds. These major new concepts largelydealing with making and controlling functional RNAs also preceded the eraof rapid DNA sequencing.

The young student of today or their youngish mentors can hardly beblamed for knowing very few details of this era, which ended before theywere born (in case of students) or before they had finished their first decadeor had begun their college years (in the case of young professors). The intel-lectual sweep of these many achievements before the early 1980s would, ofcourse, be available by reading a selected sample of the hundreds of originalpapers from 1960 to 1980. But a relatively abbreviated historical discussion

Introduction 3

told from the point of view of a long-interested RNA biochemist has beenunavailable. This is what compelled me to assemble the material in thisbook. I note here that I began working with the RNA of poliovirus in thelate 1950s in the laboratory of Harry Eagle at the National Institutes of Health.This was followed by a year (1960–1961) with Francois Jacob at L’InstitutPasteur at the time when the concept and proof of mRNA were just beingdescribed (although I had absolutely nothing to do with these landmarkexperiments). However, my own laboratory work was, and still is, directedby these early very fortuitous training experiences and perhaps will help thereader to forgive the personal voice that appears in various spots throughoutthe book.

Chapter 1 presents early discoveries that were not fitted into an under-standable fabric of cell function for decades. For example, more than 100years ago, organic chemists were able to identify all of the nucleobases,even placing uracil only in “yeast” nucleic acid (aka RNA) and thymineonly in “nuclein,” later “thymus” nucleic acid (aka DNA). However, onlyin 1920 was deoxyribose finally identified as the sugar in DNA.

The peptide bond was described and accepted as the most probable linkamong amino acids by 1902. But a long disputatious history of the molecularnature of proteins followed, literally until after World War II. How could sci-entists of 1950 begin to think of cells making proteins by uniting amino acidsin the correct order (step by step and therefore uncovering RNA functions)until after 1951, which brought Linus Pauling’s models of the a-helix andFred Sanger’s sequencing of the first chain of insulin?

The monumental accomplishments of George Beadle and Edward Tatumin showing that genes were responsible for the function of individual proteins(enzymes) and the discovery of DNA as the genetic material by Oswald Avery,Colin MacLeod, and Maclyn McCarty are stories that preceded Watson andCrick and are known by many at least in outline. But a recitation of exactlywhat experiments these heroes performed does not trip lightly off the tongueof the majority of today’s biologists, young or old.

Therefore, in diplomatic language, after “frank discussions” with my edi-tors, and with the support of many colleagues who have read early versions ofthe book, Chapter 1 presents some of this history of the centrality of macro-molecules, with my hope that, at the very least, it will be entertaining.

Chapter 2 needs no such defense. If we were going to have a history ofRNA, it was obligatory to recount the signal achievements of the 1950s–1960s that finally brought RNA out of the shadows. On reflection, viruseswith only RNA as a genome were obvious candidates to first catch attentionfor the genetic/biochemical importance of RNA. This proved to be the case,

4 Introduction

with TMV (tobacco mosaic virus) and the RNA formed after T-even (T2, T4)bacteriophage infection of Escherichia coli leading the way.

Although the gene/protein connection was made in the 1940s, it took invitro protein synthesis by rat liver extracts, largely carried out by Paul Zamec-nik and colleagues, to begin to truly connect proteins and RNA in 1953–1958. These years saw the discovery of transfer RNA (tRNA) and establisheda role for ribosomes (and presumably their RNA) in making proteins. Giventhese advances, it remains something of a puzzle as to why it took so manyremarkably gifted scientists so long after the Watson-Crick structure (�7years) to hit intellectual and molecular pay dirt with the idea of and discoveryof mRNA. This is one of the most intriguing stories in the history of molec-ular biology. The secret lay in closer attention to the genetics of gene regula-tion that explained switches in the proteins that the cell made. Making mRNAin a controlled fashion in the test tube was then accomplished with bacterialsystems in the late 1960s and early 1970s. Discussion of all these accomplish-ments constitutes Chapter 2.

Chapter 3 guides the reader stepwise through achievements that unlockedthe universal genetic code. Virtually all biology students and many from otherdisciplines will know the conclusions of this era. The aim, however, is to putsome experimental meat on the bones of the catechism symbolized by “DNAmakes RNA makes protein.” The clarity of these conclusions led GuntherStent, a physicist turned biologist, to title a 1968 paper in Science “ThatWas the Molecular Biology That Was,” in which he seemed to argue it wasall over but the shouting.

The stunning achievements on bacterial gene functions were the logicaltakeoff point for beginning to work toward understanding how eukaryoticcells controlled their genes and performed such tricks as differentiation.Chapter 4 picks up this problem that began, however, with several years ofinability to define mRNA in eukaryotic cells. Kinetic studies following incor-poration of labeled RNA precursors into properly separated classes of RNA incultured animal cells and in animal cells infected with DNA viruses finallywrestled this problem to the ground. Processing of preribosomal RNA (pre-rRNA) was uncovered by 1962–1963 and processing of pre-tRNA by 1968,but RNA processing to make mRNA was not completely understood untilthe final details of splicing of adenovirus pre-mRNA into mRNA were discov-ered in 1977. This �15-year period (1962–1977) is recounted in detail inChapter 4. Also described in this chapter are the initial discoveries of the bio-chemistry of the three eukaryotic RNA polymerases and the original illustra-tion of the complexity of nuclear factors required to initiate RNA synthesiscorrectly in cultured human cells and cell-free systems.

Introduction 5

Between the discovery of RNA splicing in 1977 and the early 1980s,additional astonishing discoveries occurred. The capacity of RNA to performenzymatic functions was recognized. Also, the involvement of previouslyunknown small ribonucleoprotein particles (in particular, their RNA) incarrying out splicing to make mRNA was discovered. These topics are alsointroduced in their appropriate historical frame in Chapter 4.

Finally, complementary DNA (cDNA) cloning and pulse-labeled nuclearRNA allowed the measurement of the rate of synthesis of individual genes,which clinched the previously widely assumed, but not yet proven, primarycontrol of gene expression at the level of transcription.

The end of Chapter 4 marks a dividing point in the book. A reasonablycomprehensive historical accounting of important events in which RNA is thechief actor ends.

Chapter 5 is an attempt to provide a useful summary of important eventsin work on RNA after the early 1980s. Because the regulation of mRNA is thecentral event in all biological specificity, a discussion of the arcane array ofproteins that control regulated transcription of chromatin is the first orderof business. The positively required factors for the initiation and manufactureof an mRNA from pre-mRNA had first to be understood. This allowed morerecent proofs of the wide variety of negative-acting proteins and protein com-plexes in preventing initiation and of the details of differential pre-mRNAprocessing, also a regulated process.

The most recent stunning advances in regulation of mRNA translationefficiency and lifetime have come from discoveries of yet more and differentRNA molecules, both short and long noncoding RNAs. How these wereuncovered and initial insight into how they function are products of researchin the last �15 years, and new discoveries and insights into noncoding RNAscontinue with each new journal issue. A running summary, necessarilyincomplete, of all this experimental activity brings up the rear of Chapter5. No attempt is made (even if it could have been) to be comprehensive inthe material of Chapter 5. Rather, important areas are included with discus-sion of some key discoveries, and up-to-date references are provided. Theshelf life of all of these new findings makes discussions about them admittedlyproblematic. But no attempt to describe a history of RNA could fail to includea digest of this recent material.

Chapter 6 is brief and highlights, first, a research area that looks back on3 billion plus years to how RNA likely had an indispensable role in initiatinglife on the planet. Organic chemical and geochemical advances are beingmade that may enlighten us about events in the Archaean era. This is possiblythe most difficult of all areas in biology, but it no longer seems the impossiblefield that it did a couple of decades ago.

6 Introduction

Perhaps of equal difficulty is the tangled problem of the origin of cells.Challenges to the conventional wisdom of prokaryote! eukaryote evolutionbegan with Carl Woese’s discovery of archaea more than 30 years ago andremains a fascinating, unsettled area today despite hundreds of genomicsequences of microorganisms. One of the most challenging unsolved prob-lems in this area also centers on RNA. How and when did splicing of RNAarise—before or after cells arose? If we had an unambiguous answer to thisquestion, might we not also better trace how the three cellular kingdoms aroseand persisted? The evolution of cells is intimately tied to the idea of an initialRNA world, a hypothetical but increasingly probable time in the evolution oflife. Given this likely history and considering the many functions of RNA inthe cells of today, shouldn’t RNA share a wedge of the spotlight with DNA?

Introduction 7

Preface

A SIZEABLE NUMBER OF COLLEAGUES READ all or portions of this book atvarious times during its preparation. Most believed, as I did, that a

book stressing the history of those phases of molecular biology centered onRNA was a sound and different idea and should produce a useful, even aneeded, book. Early discussions with Shai Shaham, Paul Nurse, Sid Strick-land, and Jan Breslow helped particularly in shaping the content of whatwas finally included. My gratitude to each of them.

As the project developed, I received positive encouragement, advice,suggestions, and corrections from a larger group. That list includes DavidAllis, Jan Breslow, Linda Chaput, Gene Cordes, Bob Darnell, Ford Doolittle,Jeff Friedman, Magda Konarska, Leon Levintow, Peter Model, Tom Muir,Paul Nurse, Lennart Philipson, Bob Roeder, Marjorie Russel, Shai Shaham,Sid Strickland, Jon Warner, and Mike Young.

To all of those friends and colleagues I offer my sincerest gratitude. Forthe remaining errors and especially for the omitted or neglected references(despite �1000 included references), the responsibility remains with me.

Initial discussions with John Inglis, the Executive Director of Cold SpringHarbor Laboratory Press, were extremely encouraging and helpful in solidify-ing the purpose of what is presented. The Cold Spring Harbor LaboratoryPress has a staff exceptionally gifted in publishing scientific works, and Iam eternally grateful to all of them: in particular, Inez Sialiano, ProjectManager, and Rena Steuer, Production Editor, who among many otherwise contributions arranged to make figures taken from older articles read-able. I especially thank Maria Smit, the Developmental Editor. I’ve had con-siderable experience in biology textbook writing and publishing but havenever had anything approaching the skillful and thoughtful editorial helpI’ve received on this book. Lois Cousseau, my assistant for more than 30 years,

ix

merits very special thanks. Nothing I’ve written in all this time, all beginning inlonghand, would have ever appeared without Lois’s cheerfulness, patience, andextraordinary competence. Even the most heartfelt thanks seem insufficient.

Finally, this labor of love was supported with my wife Kristin’s unflaggingconfidence and belief that I could do it.

x Preface

Index

Page references followed by f denote figures.

AAbelson, John, 202, 207Abrams, Richard, 171–172Adaptor hypothesis, 102Adenine, 31Adenosine triphosphate (ATP), 31, 70Alanine, 12Albert Einstein College of Medicine, 163Alexander, Hattie, 49Allfrey, Vincent, 265Allfrey-Mirsky laboratories, 157Allis, David, 266Alloway, J. Lionel, 42–43Alpher, Ralph, 101–102Altman, Sidney, 202, 204, 352a-Amanitin, 208, 210, 218Ambros, Victor, 312Ames, Bruce, 86Amino acids, 12–14, 73–75,

108–109, 353Ancient Earth, Ancient Skies: The Age

of Earth and its CosmicSurroundings, 366

Anfinsen, Christian, 72AraC gene, 124Archaean Earth. See RNA and the

beginning of lifeAcritarchs, 367Argonaute family, 321Asparagine, 12

Astbury, William T., 24Astrachan, Lazarus, 64, 66, 76, 91, 152ATP (adenosine triphosphate), 31, 70Atwood, Kim, 163Austrian, Robert, 42, 43Avery, Oswald, 4, 41–42, 43,

44–45, 47Avery, Roy, 45Ayala, Francisco, 381

BBachenheimer, Steve, 189Bacterial conjugation, 40Bacteriophages, 64–66, 81–83Baltimore, David, 175, 176Banerjee, Amiya, 180Barnett, Leslie, 109Barrell, Bart, 370Bartel, David, 321, 358Bateson, William, 34Baulcombe, David, 317Bawden, F.C., 61Beadle, George, 4, 38, 39–40, 58Beaumont, William, 12Belozersky, Andrei, 76Benjamin, Tom, 170Benner, Steve, 354Benzer, Seymour, 109, 130Berg, Paul, 74Berget, Susan, 192

399

Bernal, J.D., 24, 25Bernhardt, Deborah, 168Berzelius, Jons Jacob, 11, 22Bird, Adrian, 289Boveri, Theodor, 33Boyer, Herb, 41Brachet, Jean, 9, 10, 67Bragg, Lawrence, 19–20Brawerman, George, 172Brenner, Sydney

C. elegans work, 311, 314genetic code work, 102, 104, 109,

113–114mRNA existence contributions,

89–90, 91Bridges, Calvin, 35Britten, Roy J., 168Broker, Tom, 192, 194, 196Bronson, H.R., 26Brown, Carolyn, 325Brown, Daniel M., 58Brown, Don, 163, 211Browne, Janet, 365Brownlee, George, 370Buchner, Eduard, 22Buick, Roger, 368Burdon, Roy, 168Burley, Stephen, 258Busch, Harris, 201

CCaenorhabditis elegans

injected dsRNA triggering of mRNAsuppression, 314, 316

lin4 and lin14 and RNA:RNAregulation, 312–314

number of miRNAs in, 318programmed cell death discoveries,

311–312Cairns, John, 41Calcitonin/CGRP, 303–304CAP protein, 126–127, 129Carboxy-terminal domain (CTD),

274–275Carlson, E.A., 35Carothers, Wallace, 21Carrel, Alexis, 143–144, 148Caspersson, Torbjorn, 9, 37, 67

Catabolite repression and the CAPprotein, 126–127, 129

Cavendish laboratory, 20Cech, Tom, 202, 205, 352, 382Cellulose, 21Chambon, Pierre, 197Chang, Howard, 327Chargaff, Erwin, 48–49, 76Chase, Martha, 50Chibnall, Albert Charles, 28Chow, Louise, 192, 194Chromatin immunoprecipitation (ChIP),

267–268Chromatin-modifying factors, 285–288Chromosomes

early chemical analysis, 37–38fly genetics work in Morgan’s lab,

34–37heredity role discovery, 33–34, 35recognition of a role in heredity,

31–32Chymotrypsin, 23Ci (cubitus interruptus) proteins, 283Cinnabar, 38–39Claude, Albert, 67, 69CLIP (cross-link immunoprecipitation),

268Cohen, Stanley, 41, 58Cohesin, 274Cold Spring Harbor Symposium, 95,

104, 196Corey, Robert B., 9, 25, 58Correns, Carl, 33Cox, Ronald, 218Crane-Robinson, Colyn, 265Crick, Francis, 2, 3

genetic code work, 104, 109, 118mRNA existence contributions, 89–90paper on the adaptor hypothesis, 102papers on RNA, 352pre-double-helix work, 13, 49, 51structure of DNA and, 57theory of the adaptor, 75

Cross-link immunoprecipitation (CLIP),268

Crowfoot, Dorothy, 24CTD (carboxy-terminal domain),

274–275

400 Index

Cubitus interruptus (Ci) proteins, 283Cyclic AMP (cAMP), 126Cyclol theory, 24–25Cytosine, 30

DDalrymple, G. Brent, 366Darnell, James

adenovirus-infected cell experiments,189

cultured animal cell work, 151–152poly(A) discovery and work, 172, 173,

180, 184polyribosomes experiments, 158,

160–162recovery of long RNA molecules in

HeLa cells, 153UV transcription mapping use,

190–192work with Eagle, 149

Darnell, Robert, 304Darwin, Charles, 365Davern, Rick, 90Davidson, Norman, 193, 194Davis, Ron, 193Dawson, Martin, 42–43DBD (DNA-binding domain), 244Delbruck, Max, 45, 50, 151DeMars, Robert, 109Deoxyribose, 31, 44de Vries, Hugo, 33Dicer complex, 319Dickerson, Richard E., 18, 23Dintzis, Howard, 120–122, 158DNA

C-value paradox, 138–139definition of promoter sequences, 127,

128f, 129discovery by Miescher, 30DNA-binding domain, 244growing base of knowledge in 1950s,

57–58introns and exons in sequences,

199–200lack of appreciation of the length of,

41methylation as an epigenetic state,

290–291

mRNA manufacture control and, 246,258, 260, 283, 285

nucleic acid bases discovery, 30–32obstacles to acceptance of as the active

agent, 47–49phage school’s acceptance of,

50–51purification of deoxyribonuclease, 46sequence in related discoveries,

29–30structure discovery, 57

DNA, The Secret of Life, 50DNA-binding domain (DBD), 244Doolittle, Ford, 372Doty, Paul, 92, 153Double Helix, The, 49Double helix discovery, 2–3Dounce, Alexander, 72DRB and transcriptional elongation,

275–277D-Ribose, 31Drosha, 319Drosophila melanogaster

ChIP assays information, 268, 269Ci transcription factor, 283C value, 139Dscam, 295–296experiments showing rRNA was

copied from ribosomal DNA,163–164

eye color studies, 38–39gene regulation studies using,

245–248genetics studies in Morgan’s lab,

34–37Groucho repressor protein,

285–286heat shock genes transcription,

277–282number of miRNAs in, 318piwi gene, 322Polycomb group, 287pri mutations, 332R-loop studies using, 193–194sex determination in, 303, 304fTFIID complex, 255–256

dsRNA triggering of mRNA suppression,314, 316

Index 401

Dujon, Bernard, 382Dulbecco, Renato, 150–151, 170du Vigneaud, Vincent, 15

EEagle, Harry, 4, 147–150, 163Eagle’s Medium, 148–150Earle, Wilton R., 144–145, 148Eddy, Bernice, 170Edman protein degradation technique, 28Edmonds, Mary, 171–172Eighth Day of Creation, The, 49, 91EJCs (exon junction complexes),

306, 309Enders, John F., 146Endosymbiosis, 378–379Englesberg, Ellis, 124Enhanceosomes, 248–249, 250fEnzymology, 21–23Ephrussi, Boris, 38, 58Epigenetics and transcriptional control

DNA methylation as an epigeneticstate, 290–291

H3K4me3 directed to TSSs, 292,294–295

Polycomb and heterochromatinrepression as modifications,291–292, 293–294f

term meaning, 288–290Escherichia coli, 40–41Eukaryotic RNA

colinerarity and, 200C-value paradox, 138–139differential poly(A) and splicing,

197–199eukaryotic/bacterial divide,

200–201exploration of an mRNA function in

eukaryotes, 138first continuous cultured animal cell

line, 144–145first continuous growing human cell

culture, 145–146genes in pieces concept, 196–197hnRNA-to-mRNA processing

evidence search, 168–169introns and exons in DNA sequences,

199–200

investigation of transfer of nuclearRNA to the cytoplasm,139–142

m7 “cap” and poly(A) tails on longhnRNAs

argument against processing tomake ovalbumin mRNA,182–183

connection between HeLa cellmRNA and hnRNA, 180

potential globin mRNA precursor,181–182

use of both ends of hnRNA,180–181

m7GpppN “cap,” 178, 179f, 180methylation in mRNA, 177–178molecular steps in information

transfer, 142poly(A) tail

added to hnRNA in the cellnucleus, 173–174, 175f

discovery of, 171–172on hnRNAs, 180identification as part of polysomal

mRNA, 172–173splicing and, 197–199use in mRNA purification,

175–176polyribosomes and animal cell mRNA

discoveryHeLa cell mRNA size and base

composition determination,160–162

identification of HeLa cell mRNA,160

polyribosomes revealed throughreticulocyte extracts, 158–159

question of ribosomal density onmRNA, 157–158

processing of large nuclear RNA intomRNA

adenovirus-specific nuclear RNAand mRNA properties,183–185

discovery of RNA–RNA splicing,192–193

mapping sites of origin of adeno-virus mRNAs, 185–186

402 Index

mapping the primary transcriptprecisely, 186

nascent chain transcript mapping,186–190

R loops, 193–196UV transcription mapping,

190–192processing of tRNA precursors to

tRNA, 166, 168proof that human cell cultures could

be made, 143–144relating specific sequences in mRNA

and hnRNA, 169–170, 171freproducible growth of mammalian

cells and virusescloning of mammalian cells by

Puck, 150development of Eagle’s Medium,

148–150quantitative animal virology work

by Dulbecco, 150–151RNA chemistry after discovery of

splicingRNA-directed RNA cleavage,

202–204self-splicing RNA, 204–207spliceosome for testing snRNPs,

201–202, 203fsplicing of pre-tRNA, 207

RNA polymerases and GTFsadenovirus as the first in vitro Pol

II template, 212–214GTFs complexity recognition,

214–216identification of three enzymes,

208–210Pol III template, 211–212RNA initiation using Pol II and Pol

III, 210–211search for mRNA in cultured animal

cellsefforts to study animal RNA, 152long RNA molecules in HeLa cells

and, 153–155nature of briefly labeled RNA, 157RNA processing recognition

through ribosomal precursorRNA, 155–157

separation of pre-rRNA from hnRNAby nuclear fractionation,163–166, 167f

transcriptional control in eukaryotesbasic questions about, 216hormone-dependent increases of

specific mRNAs, 218–221run-on transcription assays and,

217–218virus production in primary cultured

animal cells, 146–147Evans, Ron, 189, 212Exon junction complexes (EJCs),

306, 309

FFerat, Jean-Luc, 383Feulgen, Robert, 67Fire, Andrew, 314, 316Fischer, Emil, 14–15, 18–19, 31Flavell, Richard, 196Flemming, Walther, 33Flint, Sarah Jane, 185Focused promoters, 241–242Formation of Colloids, The, 20Fox, George, 370Fraenkel-Conrat, Heinz, 62, 64, 108, 152Fraser, Nigel, 189Fritz-Laylin, L.K., 374Fruton, Joseph, 19

Gb-Galactosidase, 77–81, 83–86Gall, Joe, 204Gallimore, Phil, 185Gamow, George, 101–102Garen, Alan, 89, 114Garen, Susan, 89Gelinas, Rich, 192Gemmatata obscuriglobus, 380Gene expression in mammalian cells. See

Eukaryotic RNAGeneral transcription factors. See GTFs

(general transcription factors)and RNA polymerases

Genetic codechromosomes’ role in heredity

discovery, 33–34, 35

Index 403

Genetic code (continued)colinerarity of genes and proteins

problem, 130definition of promoter sequences in

DNA, 127, 128f, 129early chemical analysis of

chromosomes, 37–38first identification of a genetic disease,

103first use of word gene, 34fly genetics work in Morgan’s lab,

34–37hemoglobin studies results, 103–104,

105fhistory of ideas regarding, 101–102Mendel’s discoveries and, 32–33polyribonucleotides used to designate

codons, 110–112protein synthesis direction work by

Dintzis, 118, 120, 121f, 122protein synthesis work by Nirenberg

decision to remain at NIH, 104,106

discovery that nucleotides encodeamino acids, 108–109

techniques used to develop asystem, 106–108

regulated RNA polymerase actionrequirement, 124–125

RNA polymerase composition, 127,128f, 129

RNA polymerase discovery, 122–124search for more positive-acting

transcriptional proteinscatabolite repression and the CAP

protein, 126–127, 129cell-free gene regulation and,

126–127start and stop signals

start codon AUG and initiatortRNA, 115

termination signals and nonsensecodons, 113–115

universality of the code, 115–116sum of knowledge by end of 1960s,

130–132synthesis of ordered RNA templates by

Khorana, 112–113, 114t

triplet code proof, 109–110tRNA reading of the mRNA code

process of information transfer,118, 120f

tRNA’s anticodon loop, 116, 117f“wobble” hypothesis, 118, 119f

Geological record and early cellular lifeacritarchs, 367geochemistry and early cellular life, 368geological samples dating, 366microfossils interpreted as bacteria or

archaea, 366–367present-day geographic distribution of

life and, 365–366Georgiev, Georgii, 152, 157, 166Germ plasma theory, 33Gey, George and Margaret, 145, 147Gierer, Alfred, 62, 152, 159Gilbert, Walter, 125, 127, 199Glycine, 12Goldberg, Seth, 190Gortner, Ross, 18Gottschling, Dan, 289Graham, Thomas, 18Granboulan, Nicole, 166Grewal, Shiv, 288Griffith, Fred, 41Gros, Francois, 91, 92Groucho/TLE repression, 285–286Grunstein, Michael, 265GTFs (general transcription factors) and

RNA polymerasesadenovirus as the first in vitro Pol II

template, 212–214GTFs complexity recognition, 214–216identification of three enzymes,

208–210Pol III template, 211–212RNA initiation using Pol II and Pol

III, 210–211Guanine, 31Guo, Su, 314Gurdon, John, 163, 249

HH3K27me3, 325H3K4me3/H3K36me3, 272, 281, 292,

294–295

404 Index

H3K9me3/H3K27me3, 274, 292Hackett, Perry, 190Hall, Ben, 76, 92, 153Hamilton, Andrew, 317Hammarsten, Einar, 37, 38, 47Harris, Henry, 140–141, 155Harrison, Ross, 143Hayes, William, 40Heat shock proteins and factors, 245Hebbes, Tim, 265Hedghog pathway, 283HeLa cells, 145–146, 147, 153–155,

160–162, 180Helix-loop-helix (HLH) family,

283, 284fHemoglobin, 17, 20Heppel, Leon, 108Herpes simplex virus, 169–170, 171fHershey, Al, 50Hertwig, Oscar, 33Heterogeneous nuclear RNA (hnRNA)

DRB and transcriptional elongationand, 275–276

hnRNA-to-mRNA processingevidence search, 168–169

lncRNAs and, 324m7 “cap” and poly(A) tails on long

hnRNAsargument against processing to

make ovalbumin mRNA,182–183

connection between HeLa cellmRNA and hnRNA, 180

potential globin mRNA precursor,181–182

use of both ends of hnRNA,180–181

Poly(A) tail added to in the cellnucleus, 173–174, 175f

separation of pre-rRNA from hnRNA,163–166, 167f

Hfq (host factor for Qb), 364HIF (hypoxia-induced factor), 246Histone demethylases, 271–272Histones

acetylationactive transcription and H3K4 and

H3K36, 272

connection to active transcription,268–269, 270f

enhancers and chromatin loops,273–274

histone demethylases, 271–272histone modifications at enhancers,

272–273histone-remodeling complexes and

NFRs, 269, 271trimethylation of H3K9 and

H3K27, 274chromatin formation and

histone acetylation role intranscriptional control,265–266

nucleosome, histone tails, and thesolenoid, 263, 264

nucleosome blocking oftranscription, 264–265

modifying enzymes and complexes,266–267

History of biologydouble helix discovery, 2–3early discovery work on RNA, 3genetic code work (see Genetic code)purpose of learning about, 2

History of macromolecules before RNAbirth of enzymology

cell-free fermentation by Buchner,22

naming of catalysts and enzymes,21–22

protein crystals and, 22–23discoveries leading to RNA, 10–11DNA (see DNA)focus on proteins

discovery of amino acids as part ofproteins, 12–14

discovery of breakdown of proteinsin stomach enzymes, 12

essential amino acids identified, 13first use of term, 11–12peptide linkage work by Fischer,

14–15synthesis of peptides, 15–16

geneticschromosomes’ role in heredity

discovery, 33–34, 35

Index 405

History of macromolecules beforeRNA (continued)

early chemical analysis ofchromosomes, 37–38

first use of word gene, 34fly genetics work in Morgan’s lab,

34–37Mendel’s discoveries and, 32–33

individual genes connection toindividual proteins

enzymes/gene connection in breadmold, 39–40

eye color studies in Drosophila,38–39

gene transfer discovery usingE. coli, 40–41

lack of appreciation of the lengthof DNA, 41

mindset hindering recognition ofRNA, 9–10

phage school’s acceptance of DNA,50–51

primary protein structure experimentsby Sanger, 27–29

protein chemistry early advancesacceptance of large polymeric

molecules, 20–21chain length and molecular weight

estimates, 17colloid chemistry, 18–19physical evidence of long chain

structure, 19–20reticence to accept a long chain

structure, 18–19ultracentrifuge and Svedberg, 20

transforming principlecharacterization of pneumococcal

extracts, 42–44identification of transformation,

41–42obstacles to acceptance of DNA as

the active agent, 46–47purification of deoxyribonuclease,

46researchers’ awareness of

importance of their discovery,44–46

support of DNA as genetic materialby Chargaff, 47–49

X-ray analysis of proteins, peptides,and amino acids

a-helix andb-pleated sheets work byPauling and Lorey, 25–26, 27f

cyclol theory by Wrinch, 24–25fiber studies, 24X-ray-diffraction pictures of

crystallized globular protein, 24HLH (helix-loop-helix) family,

283, 284fhnRNA. See Heterogeneous nuclear RNAHoagland, Mahlon, 73Hofmeister, Franz, 15, 19Holley, Robert, 73, 116, 370Holoenzyme, 127Holtzman, Eric, 163Hoppe-Seyler, Felix, 30Horvitz, Robert, 312, 314, 322HOTAIR (Hox antisense inhibitory

RNA), 327–329, 330fHotchkiss, Rollin, 49, 92Hox genes, 248, 327–329Hoyrup, M., 19HP1 repression, 287–288Hsu, Ming-Ta, 196Hurwitz, Jerard (Jerry), 123Hypoxia-induced factor (HIF), 246

Iiþ gene, 84–86, 87Id proteins, 283, 284fIngram, Vernon, 75, 103–104, 116Inosinic acid, 31Insulin, 27Introns, 199–200, 382–386Itano, Harvey, 103

JJacob, Francois, 3, 4

gene control studies, 77, 79genetic code work, 124, 131gene transfer work, 40, 41mRNA hypothesis, 10, 86, 89, 352negative gene regulation work, 83

Jacob-Monod hypothesis of mRNAacceptance of mRNA’s existence and

role, 95–96consideration of an mRNA product,

87–89

406 Index

enzyme induction and lac operon,79–81

evidence for mRNA found by aCaltech group, 90–93, 94f

gene control studies using lactose,77–79

molecular hybridization techniqueused to prove mRNA, 93, 95

negative gene regulation, 83–86proposal of the existence of mRNA,

89–90, 352proposed existence of a messenger, 77protein synthesis machinery

investigation, 86–87temperate bacteriophages and the

lysogenic state, 81–83Jelinek, Warren, 189Johannsen, Wilhelm Ludwig, 34Journal of Experimental Medicine, The

(JEM), 45, 46Joyce, Gerald, 358Judson, Horace Freeland, 45, 49, 91

KKadonaga, Jim, 241Kates, Joe, 172, 173Keller, Elizabeth, 73Kelley, Dawn, 177Kemphues, Ken, 314Khorana, H. Gobind, 112–113Khoury, George, 196Kleinschmidt, A.K., 41Klessig, Dan, 192Knight, C. Arthur, 62Knopf, Paul, 158Kohne, David, 168Kolreuter, Joseph, 32Koonin, Eugene, 381, 383, 385Kornberg, Arthur, 110–112, 123Kornberg, Roger, 257, 260Kossel, Albrecht, 30–31Kunitz, Moses, 44, 46

Llac genes, 83, 84, 87Lacks, Henrietta, 145lac operon, 127–129Lactose, 77–81Langmuir, Irving, 24–25

Last universal common ancestor (LUCA),371

Latent transcription factors, 254Lateral gene transfer, 372–373Lavi, Uri, 181l Bacteriophage, 82–83Leder, Philip, 196Lederberg, Esther, 82Lederberg, Joshua, 40, 41, 47, 79Lerman, Leonard, 109Lerner, Michael, 201Levene, Phoebus, 31, 46, 48Levinthal, Cyrus, 104Lewis, Edward B., 248Likely, Gwendolyn, 144lin4 and lin14, 312–314, 321Lindberg, Uno, 170Linnaeus, Carl, 32Lipmann, Fritz, 70Lis, John, 277, 280Long-noncoding RNAs (lncRNAs)

conclusions, 332–333from defined transcription units,

326–329, 330fFlo11 and transcriptional regulation,

331hnRNA and, 324misnaming of noncoding RNAs, 332p53-dependent repression, 329RNA profiling, 325–326transcription increased by, 329Xist, 324–325

LUCA (last universal common ancestor),371

Luria, Salvador E., 51, 109Luse, Don, 212Lwoff, Andre, 77, 82Lyell, Charles, 365Lyon, Mary, 324Lysogenic state, 82–83

Mm7GpppN “cap,” 177–178, 179f,

180–183Maaløe, Ole, 89MacLeod, Colin, 4, 43, 45Macromolecule term invention, 21Magasanik, Boris, 126Maizel, Jake, 196

Index 407

Maniatis, Tom, 248Marcaud, L., 166Marcus, Philip, 150Margulis, Lynn, 378Markham, Roy, 64Marmur, Julius, 92Martin, William, 381, 383, 385Martin-Koonin model, 381–386Matthaei, Heinrich, 106, 158Mattick, John, 326Maturase, 380, 382Maxam, Allan, 127, 199Mayr, Ernst, 371McCarty, Maclyn, 4, 43–44, 45, 46,

47, 49McKnight, Stan, 181–182, 218, 219Mechanism of Mendelian Heredity,

The, 35Mediator coactivator complex, 257–258,

259f, 274Mello, Craig, 314, 316Mendel, Gregor, 32–33Mendel’s Legacy, 35Merrifield, Bruce, 15–16Meselson, Matthew, 90Michel, Francois, 382, 383Miescher, Friedrich, 29, 30Miller, Stanley, 353miR150, 323miRNAs

encoding by genomic sequenceslocation and numbers of

sequences, 317–318mRNA sites as targets for small

RNAs, 321–322Piwi-interacting RNA, 322pri-miRNA and pre-mRNA

processing, 318–321specific actions of small RNAs in

mammals, 322–324siRNAs studies involving C. elegans

andinjected dsRNA triggering of

mRNA suppression, 314, 316let7 and small RNA inhibition of

mRNA function, 314, 315flin4 and lin14 and RNA:RNA

regulation, 312–314

programmed cell death discoveries,311–312

unstable naturebacterial mRNA turnover, 309eukaryotic mRNA turnover,

310–311Mirsky, Alfred E., 25, 43–44, 46–47Missense codons, 114Miura, Kin-ichiro, 178Mizutani, Satoshi, 175MMTV (mouse mammary tumor virus),

218Monod, Jacques, 3

gene control studies, 77, 79genetic code work, 124, 131gene transfer work, 40mRNA hypothesis, 10, 86,

89, 352negative gene regulation work, 83

Montmorillonite, 369Moore, Claire, 192Morgan, Thomas Hunt, 34, 38Moss, Bernard, 178Mouse L cells, 144–145Mouse mammary tumor virus (MMTV),

218Mowshowitz, Deborah Bernhardt, 168mRNA manufacture control

ChIP assays, 267–268differential processing of pre-mRNA

cell-specific splicing factors,303–306, 307–308f

major and minor splice sitesequences and spliceosomes,299

molecular recognition mechanismsin mRNA processing,296–297

poly(A) addition complexes andsite choice, 297–299

quantities of genes involved,295–296

RNA-binding proteins’ roles, 306,309

sequences that enhance and silencesplicing, 300

spliceosome and its actions,300–302

408 Index

splice sites and splicing machineryrecognition, 299

epigenetics and transcriptional controlDNA methylation as an epigenetic

state, 290–291H3K4me3 directed to TSSs, 292,

294–295Polycomb and heterochromatin

repression as modifications,291–292, 293–294f

term meaning, 288–290histone acetylation

active transcription and H3K4 andH3K36, 272

connection to active transcription,268–269, 270f

enhancers and chromatin loops,273–274

histone demethylases, 271–272histone modifications at

enhancers, 272–273histone-remodeling complexes and

NFRs, 269, 271trimethylation of H3K9 and

H3K27, 274histone-modifying enzymes and

complexes, 266–267histones and chromatin formation

histone acetylation role intranscriptional control,265–266

nucleosome, histone tails, and thesolenoid, 263, 264

nucleosome blocking oftranscription, 264–265

lncRNAsconclusions, 332–333from defined transcription units,

326–329, 330fFlo11 and transcriptional

regulation, 331hnRNA and, 324misnaming of noncoding RNAs,

332p53-dependent repression, 329RNA profiling, 325–326transcription increased by, 329Xist, 324–325

miRNA encoding by genomicsequences

location and numbers ofsequences, 317–318

mRNA sites as targets for smallRNAs, 321–322

Piwi-interacting RNA, 322pri-miRNA and pre-mRNA

processing, 318–321specific actions of small RNAs in

mammals, 322–324miRNAs and siRNAs studies involving

C. elegansinjected dsRNA triggering of

mRNA suppression, 314, 316let7 and small RNA inhibition of

mRNA function, 314, 315flin4 and lin14 and RNA:RNA

regulation, 312–314programmed cell death discoveries,

311–312mRNAs’ unstable nature

bacterial mRNA turnover, 309eukaryotic mRNA turnover,

310–311processing decisions discoveries, 236recent progress in eukaryotic gene

expression, 333–334regulated transcriptional events after

initiationDRB and transcriptional

elongation, 275–277locating transcription start sites,

281–282paused polymerases and elongation

step, 277–280phosphorylation of Pol II CTD,

274–275short promoter-proximal

transcripts, 280–281regulation discoveries, 236–237siRNAs in plants, 317summary of regulation of gene

expression, 234ttechnological advances contributions,

238–239transcriptional activators and

initiation

Index 409

mRNA manufacture control (continued)activator proteins’ functional

domains, 244–245cell signaling: external signaling

proteins, 252, 253f, 254cell signaling: latent transcription

factors, 254cell signaling: steroid receptor

superfamily, 251, 252fdevelopmental and cell-specific

functions, 246–248DNA damage and p53 activation,

246enhanceosomes, 248–249, 250fMediator coactivator complex,

257–258, 259f, 274numbers and function, 243–244response to environmental stress,

245–246in stem cells, 249TBP–DNA–TFIIB structure, 258,

260TFIID and TAFs, 255–257yeast structure determination

through crystallography,260–263

transcriptional initiation regulation ofpre-mRNA

enhancers and discovery of tran-scriptional activator proteins,242–243

locating promoters, 239–242transcription blocking in eukaryotic

cellsdirect-acting repressor proteins,

282–283, 284finhibition of the action of a

DNA-bound activator, 283, 285transcriptional inhibition by

chromatin-modifying factors,285–288

transcription field discoveries,235–236

Mulder, Gerrit Jan, 11Muller, Hermann, 35, 37Muller, H.J., 35Muller-Hill, Benno, 125My Life in Science, 91

NNakajima, N., 255National Institutes of Health (NIH),

104, 112Neurospora crassa, 39–40Nevins, Joe, 190NFRs (nucleosome-free regions), 269,

271NICD (Notch intracellular domain), 244Nieman, Carl, 25NIH (National Institutes of Health), 104,

112Nirenberg, Marshall, 3, 104, 106,

112, 158NMD (nonsense-mediated decay), 309Nobel: The Man and His Prizes, 47Noll, Hans, 159Noller, Henry, 359Nomura, Masayasu, 76, 92Nonsense codons, 114Nonsense-mediated decay (NMD), 309Northrop, John, 23, 44, 60Notch intracellular domain (NICD), 244Nova proteins, 304–306, 307–308fNuclear receptors (NRs), 251Nucleic acid bases discovery, 30–32Nuclein, 30–31Nucleosome-free regions (NFRs), 269,

271Nucleosomes, 263, 264–265Nusslein-Volhard, Christiane (Jani), 246

OOchoa, Severo, 110–112Orgel, Leslie, 89, 352, 356, 358Ørom, Ulf, 329Osborne, Thomas, 17

PP1 RNase, 204p53 activation, 246Pace, Norman, 203–204Painter, Theophilus S., 35PaJaMa experiment, 83–86, 87, 106Palade, George, 68Palmiter, Richard, 218, 219Pardee, Arthur, 83–84, 87Pastan, Ira, 126

410 Index

Pauling, Linus, 4, 9, 25–26, 27f, 58, 103Payne, Fernandus, 34Penicillin, 148–150Penman, Sheldon, 160, 163Penman-Holtzman nuclear fractionation

technique, 163–166, 167fPenny, David, 385Pentose nucleic acid (PNA), 10Pepsin, 23, 60Peptides, 12, 14–16, 24–26, 27fPeptidyl synthesis by rRNA, 359–361Perry, Robert, 152, 155, 163, 177Phaseolus vulgaris, 34Philipson, Lennart, 183–185Phosphate, 31Pirie, N.W., 61Pisum sativum, 32Piwi-interacting RNAs (piRNAs), 322Planctomycetes, 380Plasmids, 41PNA (pentose nucleic acid), 10Pol II and Pol III, 210–214, 255, 274–275Poliovirus vaccine, 146–147Poly(A) tail (polyadenylic acid)

added to hnRNA in the cell nucleus,173–174, 175f

addition complexes and site choice,297–299

differential poly(A) and splicing,197–199

discovery of, 171–172on hnRNAs, 180identification as part of polysomal

mRNA, 172–173splicing and, 197–199use in mRNA purification, 175–176

Polycomb repression, 287, 291–292,293–294f, 325

Polynucleotide phosphorylase, 111–112,113

Pontecorvo, Guido, 200–201Poole, Anthony, 385Porter, Keith, 68Positive-acting transcriptional proteins,

124–125Present at the Flood, 23pri mutations, 332Principles of Geology, 365

Prophage, 82Protein synthesis and RNA

cell fractionation, 67–68cell-free extracts and, 69–70Crick’s theory of the “adaptor,” 75cytoplasmic RNA related to protein

synthesis, 67directional work, 118, 120, 121f, 122discovery that nucleotides encode

amino acids, 108–109observation by electron microscope,

68ribosomes named and accepted as the

site of protein synthesis, 70, 71fsearch for the missing piece of mRNA,

75–76techniques used to develop a system,

106–108template hypothesis, 71–72tRNA and amino acid–activating

enzymes discovery, 73–75Protoeukaryote, 381Ptashne, Mark, 125Puck, Theodore, 150Pyrimidines, 30

RRajewsky, Klaus, 324Reannealing, 92Reddy, Ramachandra, 201Reichard, Peter, 47Reverse transcriptase, 176Ribozymes, 358Rich, Alexander, 158Riley, Monica, 87RISC (RNA-induced silencing complex),

320–321Ritossa, Ferruccio M., 163R loops, 193–196RNA and the beginning of life

archaean2a-proteobacterial unionprecursors to introns, 382–386proposal bases, 381–382

central roles of RNA in macromolec-ular synthesis, 352

development of a precellular state, 386eukaryotic cells origin

endosymbiosis, 378–379

Index 411

RNA and the beginning of life (continued)possible evolutionary pathways to

an eukaryote, 379–381first informational molecule,

351–352geological record and early cellular

lifeacritarchs, 367geochemistry and early cellular life,

368geological samples dating, 366microfossils interpreted as bacteria

or archaea, 366–367present-day geographic

distribution of life and,365–366

prebiotic chemistryamino acids, 353nucleotides, 353–356

present-day RNA machines and anRNA world

peptidyl synthesis performed byrRNA, 359–361

pre-rRNA processing andsnoRNAs, 361, 362f

RNase P1, 361, 363signal recognition particle, 363small RNAs and, 364–365telomerase, 363–364

primacy of RNA, 387–388protocell research, 368–369self-replication of RNA,

356–359three kingdoms of life proposal by

Woese, 369–371whole-genome sequencing and origin

of cellular kingdomsarchaeal–eurkaryotic similarity,

374, 376–378lateral gene transfer, 372–373protein sequence comparisons and

evolutionary history, 373–374,375f

RNA as the connector of genes andproteins

growing base of knowledge of DNA in1950s, 57–58

Jacob-Monod hypothesis of mRNA

acceptance of mRNA’s existenceand role, 95–96

consideration of an mRNAproduct, 87–89

enzyme induction and lac operon,79–81

evidence for mRNA found by aCaltech group, 90–93, 94f

gene control studies using lactose,77–79

molecular hybridization techniqueused to prove mRNA, 93, 95

negative gene regulation,83–86

proposal of the existence ofmRNA, 89–90

proposed existence of a messenger,77

protein synthesis machineryinvestigation, 86–87

temperate bacteriophages and thelysogenic state, 81–83

research leading to hypothesis of anRNA role in gene regulation,58–60

RNA’s role in protein synthesiscell fractionation, 67–68cell-free extracts and, 69–70Crick’s theory of the “adaptor,” 75cytoplasmic RNA related to protein

synthesis, 67observation by electron micro-

scope, 68ribosomes named and accepted as

the site of protein synthesis, 70,71f

search for the missing piece ofmRNA, 75–76

template hypothesis, 71–72tRNA and amino acid–activating

enzymes discovery, 73–75RNA viruses

bacteriophage infection and,64–66

determination of the basecomposition of RNA, 66

importance of timing in researchdiscoveries, 64

412 Index

proof of the genetic role of TMVRNA, 62–64

TMV studies, 60–62viruses’ genetic capacity

understanding, 60state of understanding of RNA in early

1950s, 58RNA-induced silencing complex (RISC),

320–321RNA polymerases

composition, 127, 128f, 129discovery, 122–124GTFs and

adenovirus as the first in vitro PolII template, 212–214

GTFs complexity recognition,214–216

identification of three enzymes,208–210

Pol III template, 211–212RNA initiation using Pol II and Pol

III, 210–211regulated action requirement

lac and l repressor proteinspurification, 125

positive-acting transcriptionalproteins and, 124–125

Robbins, Frederick, 146Roberts, Richard, 70, 197The Rockefeller Institute, 67, 68, 92,

157, 196Rodrıguez-Trelles, Francisco, 381Roeder, Robert G., 208, 211, 212, 214,

255, 264Roizman, Bernard, 169Rose, William C., 13Rothen, Alexandre, 44Rubber, 21Rutter, W.F. “Bill,” 208Ruvkun, Gary, 312, 314

SSalditt-Georgieff, Marianne, 180Salk, Jonas, 146Sandford, Katherine, 144Sanger, Fred, 4, 9, 27–29, 58, 199Sarabhai, Anand, 130Sauerbier, Walter, 190

Schaffner, Walter, 242Scherer, William, 147Scherrer, Klaus, 152, 153, 160, 166Schimke, Bob, 182, 197Schleiden, Matthias, 33Schleif, Robert, 124Schramm, Gerhard, 62, 152Schultz, Jack, 38Schwann, Theodor, 12, 33Sebe-Pedro, A., 374Second messengers, 254Seed sequences, 321Segall, Jackie, 212Sevag, M.G., 43Sharp, Phil, 185, 192, 194, 196, 197Shatkin, Aaron, 178, 180Short inhibitory RNA (siRNA), 317Short RNAs, 280–281Shubin, Neil, 365Sickle cell anemia, 103Siekevitz, Philip, 68, 69Sigma (s) factor, 127Signal receptor domains, 244Signal recognition protein (SRP), 363Singer, Maxine, 108siRNA (short inhibitory RNA)

and miRNAs studies involvingC. elegans

injected dsRNA triggeringof mRNA suppression,314, 316

let7 and small RNA inhibition ofmRNA function, 314, 315f

lin4 and lin14 and RNA:RNAregulation, 312–314

programmed cell death discoveries,311–312

in plants, 317Smadel, Joseph, 109Sm proteins, 364, 385snRNAs, 300–302snRNPs, 201SOCS1 (suppressor of cytokine

signaling 1), 323Soeiro, Ruy, 164Sørenson, S.P.L., 19, 20Spiegelman, Sol, 76, 92, 163Spirin, Alexander, 76

Index 413

Spliceosomesactions of, 300–302impact of discovery of, 382–386mRNA manufacture control and,

297–299for testing snRNPs, 201–202,

203fSRP (signal recognition protein), 363Stahl, Frank, 90Stanley, Wendell, 58Start and stop signals

start codon AUG and initiator tRNA,115

termination signals and nonsensecodons, 113–115

universality of the code,115–116

Statue Within, The, 83, 89Staudinger, Hermann, 21Steinberg, Daniel, 72Steitz, Joan, 201, 299Stem cells and transcriptional activators,

249Stent, Gunther, 5, 131Steroid receptor superfamily,

251, 252fStevens, Audrey, 123Stewart, Sarah, 170St. Martin, Alexis, 12Stretton, Tony, 130Stromatolites, 366“Studies on the Chemical Nature of

the Substance InducingTransformation ofPneumococcal Types,” 45

Sturtevant, Alfred, 35Sulston, John, 311Summons, Roger, 368Sumner, James B., 22, 60Suppression/suppressor

strains, 114Suppressor of cytokine signaling 1

(SOCS1), 323Sutherland, Earl, 126Sutherland, John, 354, 387Sutton, Walter S., 33, 34SV40 virus, 169–170, 171fSvedberg, Theodor (The), 20

Syverton, Jerome, 147Szostak, Jack, 358, 369

TTAD (transcription activation

domain), 244TAFs (TBP-associated factors), 255Tarrıoa, Rosa, 381Tata, J.R., 218Tatum, Edward L., 4, 39–40, 58Taylor, Harriet, 49TBP-associated factors (TAFs), 255Telomerase, 363–364Temin, Howard, 175, 176Temperate bacteriophages, 82Tetrahymena thermophila, 204–207, 266,

356–358, 382Tetranucleotide hypothesis, 31TFIID and TAFs, 255–257, 258, 260Thompson, E.O.P., 28Thorne, Alan, 265Three kingdoms of life proposal by

Woese, 369–371Thymine, 30, 31Thymus nucleic acid, 31Tiktaalik, 365Tissieres, Alfred, 91, 106Tjian, Robert, 255TLE repression, 285–286Tobacco mosaic virus (TMV) and RNA,

58–59, 60–64, 108Todd, Alexander R., 58Tomkins, Gordon, 106Tonegawa, Susumu, 170Transcription

activators and initiationactivator proteins’ functional

domains, 244–245cell signaling: external signaling

proteins, 252, 253f, 254cell signaling: latent transcription

factors, 254cell signaling: steroid receptor

superfamily, 251, 252fdevelopmental and cell-specific

functions, 246–248DNA damage and p53 activation,

246

414 Index

enhanceosomes, 248–249, 250fMediator coactivator complex,

257–258, 259fnumbers and function,

243–244response to environmental stress,

245–246in stem cells, 249TBP–DNA–TFIIB structure, 258,

260TFIID and TAFs, 255–257yeast structure determination

through crystallography,260–263

blocking in eukaryotic cellsdirect-acting repressor proteins,

282–283, 284finhibition of the action of a

DNA-bound activator,283, 285

transcriptional inhibition bychromatin-modifying factors,285–288

field discoveries, 235–236initiation regulation of pre-mRNA

enhancers and discovery oftranscriptional activatorproteins, 242–243

locating promoters, 239–242Transcription activation domain (TAD),

244Transcription start site (TSS), 235,

281–282, 292, 294–295Transesterification, 205Transforming principle

characterization of pneumococcalextracts, 42–44

identification of transformation,41–42

obstacles to acceptance of DNA as theactive agent, 46–47

purification of deoxyribonuclease, 46researchers’ awareness of importance

of their discovery,44–46

support of DNA as genetic material byChargaff, 47–49

Transforming Principle, The, 47

tRNAamino acid–activating enzymes

discovery and, 73–75processing of tRNA precursors to

tRNA, 166, 168reading of the mRNA code

process of information transfer,118, 120f

tRNA’s anticodon loop,116, 117f

“wobble” hypothesis, 118, 119fsplicing of pre-tRNA, 207start codon AUG and initiator, 115

Trypsin, 23Tschermak-Seysenegg, Erich, 33TSS (transcription start site), 235,

281–282, 294–295Tuppy, Hans, 28Turner, Chris, 265Turnip yellow mosaic (TYM), 64

UUnfolded protein complexes, 245Uracil, 31Urease, 22, 60UV transcription mapping,

190–192

VVermillion, 38–39Verrucomicrobia, 380Vinograd, Jerome, 90Vogt, Marguerite, 151Volkin, Elliot, 64, 66, 76, 91, 152von Ehrenstein, Gunter, 116

WWaddington, C.H., 289Wagner, Ed, 169Warner, Jonathan, 158, 163Watson, James D., 2

mRNA existence contributions, 89pre-double-helix work, 13, 49, 50structure of DNA and, 57

Weber, Jeffrey, 189Wecker, Eberhard, 153Wei, Cha-Mer, 178

Index 415

Weil, Tony, 212Weiss, Sam, 123Weissman, August, 33Weissman, Sherman, 370Weller, Thomas, 146Westphal, Heiner, 196White, Ray, 193Widnell, C.C., 218Wieschaus, Eric, 246Wild, Martha, 204Wilkins, Maurice, 51Willard, Huntington, 325Williams, Robley, 62Willstatter, Richard, 23Wilson, Edmund B., 31–32, 34Wilson, Michael, 189Witkowski, J.A., 144“Wobble” hypothesis, 118, 119fWoese, Carl, 7, 352, 369–371, 377,

381, 386

Wollman, Elie, 40, 79, 82, 83Wrinch, Dorothy, 24–25

XX-Inactivation center (Xic), 325X-Inactivation RNA (Xist), 324–325X-Ray sensitivity of cells, 150

YYanofsky, Charles, 130Yeast nucleic acid, 31Young, Richard, 257

Zzþ gene, 84–86, 87Zamecnik, Paul, 5, 69, 73Zaug, Arthur, 205Ziff, Ed, 189, 212Zymase, 22

416 Index