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The FASEB Journal Book Review

RNA: Lifes Indispensable Molecule by James Darnell (2011) CSHL Press


Robert Haselkorn1
Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois, USA Jim Darnells career in science covers the 60 or so years following the publication of the Watson-Crick structure of DNA. This remarkable book tells a story that parallels his career, dealing at the beginning with the prehistory of research on RNA, DNA, and proteins and then shifting into high gear with a detailed look at the history of bacterial messenger RNA and the authors own specialty, the RNA of eukaryotic cells. Having been present at the creation, so to speak, and being blessed with an incredible memory (or really good les), Darnell is able to produce an accurate ow, naming the right names and supporting every assertion with examples of good data from the historic record. The prehistory of Chapter 1 ends with the structure of DNA in 1953. The second chapter describes the discovery of messenger RNA in bacteria. From my perspective, Darnell gets this just right. I was a postdoc in Roy Markhams lab in Cambridge from 1959 1961, just outside the city but able to see Francis Crick and Sydney Brenner often, and friendly with Jim Ofengand, who was in the Brenner lab. Brenner went off to Cal Tech in the summer of 1960 with the express purpose of using the CsCl equilibrium centrifugation technique developed by Matt Meselson to see whether Volkin RNA in T2-infected E. coli could be found associated with old ribosomes, that is, ones that existed before T2 infection. E. Volkin and L. Astrachan worked at Oak Ridge, where 32 P was available readily. By 1956, they had used the label to study nucleic acid metabolism in E. coli infected with phage T2. They found that early in infection an unstable RNA could be labeled rapidly with 32P and that the label turned over and ended up in T2 DNA. The base composition of the RNA resembled that of the phage DNA. Here is where their relative isolation in Oak Ridge cost them: they interpreted their result to mean that the RNA was a precursor of the DNA. Never mind that it would require converting every ribose to deoxyribose and methylating every U to T. I suspect that it was this erroneous interpretation that caused others to ignore the results themselves,
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which were absolutely correct. Sol Spiegelman proposed another reason the Volkin result was ignored: he said it was because it had not been predicted by Francis. Whatever the reason, it was ignored until the Brenner, Jacob, and Meselson experiment in 1960, when it was essentially conrmed and re-interpreted to mean that the Volkin RNA was a message carrying the gene sequence to the ribosome, where it could be translated (somehow) into the amino acid sequences of the phage proteins. Darnell is an experienced teacher and author of textbooks. His explanations of complex experiments are superb, taking, for example, his description of the phage genetic experiments in which Crick himself participated, the ones that established the triplet nature of the genetic code. These experiments involved careful analysis of the rII region of phage T4, in which chemical agents were used to induce and then to revert deletion or insertion mutations. Darnells recapitulation of these, from motivation to outcome, is a paradigm of clarity. A dicey aspect of the historical approach is the assignment of credit, which occasionally requires disputing the judgment of others, such as the Nobel committee. I nd Darnell to be right on target in these cases, perhaps even too gentle in some. One example is the prize for the discovery of the enzyme that synthesizes RNA, awarded to Severo Ochoa. The committee was completely misled, believing that polynucleotide phosphorylase was the real RNA polymerase, when in truth it degrades RNA. The real RNA polymerase was rst isolated by Sam Weiss and then by Jerry Hurwitz and Audrey Stevens, which Darnell correctly emphasizes.

1 Correspondence: Dept. of Molecular Genetics and Cell Biology, University of Chicago, 920 E. 58th St., CLSC, Rm. 1039A, Chicago, IL 60637, USA. E-mail: r-haselkorn@uchicago.edu doi: 10.1096/fj.11-1101ufm

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I want to return to the matter of polynucleotide phosphorylase. That enzyme was discovered by Marianne Grunberg-Manago when she was a visitor in Ochoas lab. Francis Crick lectured in a graduate course at Harvard in 19571958. He devoted one talk to the enzyme, mentioning its discovery and noting that ever since, it had been known as the Ochoa enzyme. For the students, he drew the conclusion that to get ahead in molecular biology, it was useful to have a simple name (pause) like Watson. Jim, in the audience, blushed. Everyone roared. And there is more. Ochoa got the prize in 1959. The following New Years Eve, my wife and I were in Mariannes apartment in Paris. Marianne had written to Ochoa to congratulate him on the prize. In return, he sent an engraved card thanking her for her congratulations. Said she: He might have sent me a rose Any history of RNA has to include the plant viruses. Here, Darnell does not disappoint, mentioning the work of Bawden and Pirie on TMV that showed it to contain some RNA and later of Gierer proving that the RNA was the infectious component. There is also an interesting paragraph about Roy Markham, the plant virologist who separated the empty protein shells of turnip yellow mosaic virus from the complete virus that contained 40% by weight as RNA. Roy found the shells to be incapable of infecting plants, while of course the complete virus could. Why did he not conclude that the RNA carried the information for virus replication? When I arrived in Roys lab as a postdoc in 1959, one of the rst things I was told by his lieutenant, Maurice Rees, was not to ask that question. But Roy adapted and he strongly supported our goal of relating the nucleotide sequence of the RNA to the amino acid sequence of the viral proteins. With Jim Ofengand, we were making progress on the TYMV-RNA directed synthesis of protein in a cell-free system from E. coli when Marshall Nirenberg blew us out of the water with his discovery of the poly(U) directed synthesis of polyphenylalanine. Blame that pesky polynucleotide phosphorylase again. Another often overlooked contributor, whose name pops up in these pages, is Ben Hall, who determined the correct size of eukaryotic ribosomal RNA while a student in the lab of Paul Doty. He then went on to Urbana where he invented DNA/RNA hybridization and next, with Masayasu Nomura, found mRNA in T2-infected E. coli. The latter work used simple centrifugation to show the association of Volkin RNA with ribosomes. Unfortunately, the Nomura, Hall, and Spiegelman paper included a second model consistent with the data, namely that T2 directed the synthesis of specic ribosomes (added at Speigelmans insistence) and that is the model Brenner chose as his straw man to knock down in the paper with Jacob and Meselson. Darnells third chapter describes the coding wars (Nirenberg vs. Ochoa) well and then nicely covers everything you want to know about the regulation of gene expression in bacteria. Aside from his participation in the famous PaJaMa experiment in Paris, the person often overlooked in discussions of the operon concept is Art Pardee. But Arts work with his student Monica Riley is given correct and fair emphasis. With regard to the nature of the repressor, Darnell mentions the role of Leo Szilard as gady in a footnote but omits Leos design of the chemo3754 Vol. 25 November 2011

stat and its use by Aaron Novick to show that the regulation of the lac operon is negative (i.e., that induction corresponds to the relief of repression). He ends the chapter with a look at Gunther Stents famous peroration, predicting the end of hope for new physical principles to be found in biology. Darnells own work is on eukaryotic cells and viruses. The fourth chapter covers gene expression in mammalian cells, where he is in his element, to put it mildly. I cannot think of anyone with greater range of knowledge and mastery of detail. This chapter includes a great deal of data from Darnells own papers, dense but always serving a heuristic purpose. An opportunity for bias is avoided deftly by crediting the discovery of polysomes to three groups: Jon Warner and Paul Knopf at MIT, Gierer in Germany (both using reticulocytes), and Hans Noll in Pittsburgh, using rat liver. In fact, the three groups published within a month or two of each other, and all three trailed the experiment of Wally Gilbert by a very short time, who found that poly(U) added to E. coli ribosomes created polysomes. This chapter becomes rather dense with data. We see how HeLa cell RNA is labeled in short-term experiments and how with great care the author and his friends determined the pathway by which pre-ribosomal RNA is processed, the nature of the heterogeneous nuclear RNA that was not ribosomal, its relationship to the true messenger RNA found in polysomes, and eventually how that RNA is modied. One of the modications, the addition of poly(A) to the 3 end of both hnRNA and mRNA, is considered in detail. The experiments of Shatkin and Moss leading to the structure of the 5 cap are given. The extensive work with viruses (polyoma virus, SV40, reovirus, vaccinia, herpes simplex, and the queen of viruses in this story, adenovirus) is presented, culminating in the discovery of splicing. The splicing story is given in superb fashion, giving historical credit to Norman Davidson, in whose Cal Tech lab the principals (Phil Sharp, Tom Broker, and Louise Chow) were trained. Their work at Cold Spring Harbor, using innovative electron microscopy of adenovirus DNA/RNA hybrid molecules, was enabled by earlier work from Darnells own lab and studies on restriction enzymes provided by Rich Roberts at Cold Spring Harbor. From this crowd, the Nobel committee singled out Sharp and Roberts for the prize; Darnell does not mention that he might have (or should have) been included, along with Broker, Chow, and Phil Leder, and Pierre Chambon. Too many angels dancing on the head of that pin. Darnells observations, almost as asides, are wonderful. He points out, for example, that the results of genomic DNA sequencing, made possible after 1977 with the introduction of Maxam-Gilbert chemical sequencing and then the chain-termination method from Fred Sangers lab, would have led to utter confusion if the EM results that led to the understanding of RNA splicing had not been obtained prior to the DNA sequencing. Processing in two systems, the conversion of pre-tRNA to mature tRNA and the removal of introns from premRNA, is presented, with emphasis on the chemical mechanisms. These studies, by Sid Altman (and Norman Pace) on tRNA and by Tom Cech on the ribosomal RNA of Tetrahymena, led to the discovery of the catalytic activity
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of RNA itself. The removal of introns from pre-tRNA, particularly in Archaea, is given short shrift in a single paragraph that mentions John Abelson but neglects the large contribution from the school of Glauco TocchiniValentini in Rome. Perhaps this neglect stems from the fact that these reactions are catalyzed by ordinary (or not so ordinary, because they recognize unique secondary structures in the introns) proteins. Speaking of not so ordinary proteins, the remainder of Chapter 4 is devoted to eukaryotic RNA polymerases. We left Sam Weiss and others in the 1960s unable to solubilize the enzyme. Weiss actually had a skilled postdoc, Shutsung Liao, who succeeded in that task. But Liao was persuaded by a local force, Charles Huggins, to switch to working on the androgen receptor. Without his help, Weiss turned to the bacterial enzyme, choosing Micrococcus lysodeikticus instead of the better characterized E. coli as the source. With students Fred Fox, Bill Robinson, Mas Nakamoto, and John Moohr and collaborating with Peter Geiduschek, Weiss made numerous and signicant contributions to the transcription eld that are largely overlooked. Eventually, the breakthrough of solubilization and fractionation leading to the identication of the three activities, Pol I, Pol II and Pol III, was achieved by Robert Roeder, then a student of Bill Rutter. More details from the Roeder lab follow, including the ow chart for purication of the general transcription factors, TF IIA, TF IIB and TF IID. The structure of the transcription factors and of RNA polymerase is postponed until the next chapter. This chapter is avowedly the last of the historical ones, since from 1980 onward the eld has exploded so rapidly that it is inconvenient to attempt to follow the same historical line better just to keep up with the facts. A small cavil: Steve McKnight is called Stan, not good in a history. There is one more such error in the nal chapter. The monumental Harry Noller, who showed that the RNA of the ribosome does all the work, is called Henry. Chapter 5, fully a quarter of the book, is densely packed with information about the ways in which mRNA is controlled in eukaryotes. Fortunately, we begin with a summary table that lists all the ways that gene expression is regulated in eukaryotes. Then, considered in order, we have the location of promoters, enhancers, transcriptional activators (including enhanceosomes, activators in stem cells, cell signaling steroid receptors, and latent transcription factors), then coactivators, TF IID and TAFs, and nally Mediator. A very handy table compares the protein components of the yeast, human and Drosophila TBP-associated factors of the TF IID coactivator complex. Finally, we see the structure of yeast Pol II and of the enzyme in the act of transcription. This work is shown in Figure 511, comprised of parts taken from three papers from the lab of Roger Kornberg. Given the signicance of this work, I found the gure too small and too hard to follow; it deserves much more space. In contrast, the

nucleosome structure by Tim Richmond on the following page is much easier to see. The discussion of chromatin and its modication that follows is comprehensive. The gures here are spotty: the drawings are mostly excellent but Figure 519 showing real data (paused polymerases revealed by uorescent probes that detect specic premRNAs) are almost invisible to these old eyes. Repression and epigenetic modications are also handled well, and are well-illustrated, except for Figure 523, which needs some editing. It shows repair of hemimethylated DNA following replication, but the methyl groups are sprinkled almost randomly, some landing correctly on C but others hit G or even the phosphate. This chapter continues with a discussion of the processing of pre-mRNA, including splicing and poly(A) addition. This is a very fast-moving eld and even with many references to work published in 2010 and 2011, some conclusions will have to be modied. Nevertheless, the information content here is so dense that it is impossible to open to any page without learning something new. How many readers know that the Dscam gene of Drosophila has 95 exons and can be spliced differentially to yield more than 30,000 different proteins from the same premRNA sequence? Or that the choice of alternative poly(A) sites on the same pre-mRNA can yield calcitonin in the thyroid or CGRP in neurons? Or that the RNAbinding proteins called Nova are responsible for cellspecic splicing in the brain, resulting in specic proteins found in synapses? The latter work is from the lab of Robert Darnell, the authors son, who clearly followed the advice Jim offered to anyone who listened: study neurobiology. The nal part of this large chapter discusses small RNAs, returning briey to the historic approach by describing the work with the nematode by Brenner and Sulston, then Horvitz, then Ruvkun and Ambros that led to the understanding of lin4 and lin14, whose RNA:RNA interaction was found to be regulatory. This discovery was followed by Ruvkuns nding that the let7 gene of the nematode, another regulator consisting of small RNA, had homologues in all animals! Next, Craig Mello and Andrew Fire injected double-stranded RNA into nematodes and observed signicant inhibition of expression of the protein whose mRNA corresponded to the injected RNA sequence. Their work led to RNAi and siRNA which, Darnell observes, opened the oodgates by permitting those working with eukaryotic cells to knock down any protein at will. The chapter includes much more that deserves careful reading. And it ends with a wistful peroration: Those of us among the thousands of us who did not discover the structure of DNA, the universal genetic code, or the rst proteins that can and do control genes can still justiably enjoy our achievements and be secure in the knowledge that we took part in a great and continuing enterprise. Amen.

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BOOK REVIEW: RNA: LIFES INDISPENSABLE MOLECULE 3755