The FASEB Journal • Book Review
RNA: Life’s Indispensable Molecule
by James Darnell (2011)
CSHL Press
Robert Haselkorn1
Department of Molecular Genetics and Cell Biology, University of
Chicago, Chicago, Illinois, USA
Jim Darnell’s 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 author’s 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 files), Darnell is able to produce
an accurate flow, 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 messen-
ger RNA in bacteria. From my perspective, Darnell gets
this just right. I was a postdoc in Roy Markham’s 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 cen-
trifugation 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 compo-
sition 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 interpre-
tation 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. What-
ever the reason, it was ignored until the Brenner, Jacob,
and Meselson experiment in 1960, when it was essentially
confirmed 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 text-
books. 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. Darnell’s recapitulation of these, from motiva-
tion to outcome, is a paradigm of clarity.
A dicey aspect of the historical approach is the assign-
ment of credit, which occasionally requires disputing the
judgment of others, such as the Nobel committee. I find
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 first isolated by Sam Weiss and then
by Jerry Hurwitz and Audrey Stevens, which Darnell
correctly emphasizes.
1
Correspondence: Dept. of Molecular Genetics and Cell Biol-
ogy, 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
3753
 I want to return to the matter of polynucleotide phos-
phorylase. That enzyme was discovered by Marianne
Grunberg-Manago when she was a visitor in Ochoa’s lab.
Francis Crick lectured in a graduate course at Harvard in
1957–1958. He devoted one talk to the enzyme, mention-
ing 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 Year’s Eve, my wife and I were in Marianne’s apart-
ment in Paris. Marianne had written to Ochoa to congrat-
ulate 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 Roy’s
lab as a postdoc in 1959, one of the first 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 poly-
phenylalanine. Blame that pesky polynucleotide phos-
phorylase 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 specific ribosomes (added at
Speigelman’s insistence) and that is the model Brenner
chose as his straw man to knock down in the paper with
Jacob and Meselson.
Darnell’s third chapter describes the coding wars (Ni-
renberg 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
Art’s 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
gadfly in a footnote but omits Leo’s design of the chemo-
3754 Vol. 25 November 2011 The FASEB Journal 䡠 www.fasebj.org
stat and its use by Aaron Novick to show that the regula-
tion 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 Stent’s famous peroration,
predicting the end of hope for new physical principles to
be found in biology.
Darnell’s 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 Darnell’s 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 mes-
senger RNA found in polysomes, and eventually how that
RNA is modified. One of the modifications, 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 discov-
ery 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 micros-
copy of adenovirus DNA/RNA hybrid molecules, was
enabled by earlier work from Darnell’s 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.
Darnell’s 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 Sanger’s
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 pre-
mRNA, 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
HASELKORN
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 Tocchini-
Valentini 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, Shut-
sung 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 Micrococ-
cus 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 significant con-
tributions to the transcription field that are largely over-
looked. Eventually, the breakthrough of solubilization
and fractionation leading to the identification 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 flow chart for
purification 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 field 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
final 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, transcrip-
tional activators (including enhanceosomes, activators in
stem cells, cell signaling steroid receptors, and latent
transcription factors), then coactivators, TF IID and TAFs,
and finally 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 5–11, comprised of parts taken from three papers
from the lab of Roger Kornberg. Given the significance of
this work, I found the figure too small and too hard to
follow; it deserves much more space. In contrast, the
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BOOK REVIEW: RNA: LIFE’S INDISPENSABLE MOLECULE
nucleosome structure by Tim Richmond on the following
page is much easier to see. The discussion of chromatin
and its modification that follows is comprehensive. The
figures here are spotty: the drawings are mostly excellent
but Figure 5–19 showing real data (paused polymerases
revealed by fluorescent probes that detect specific pre-
mRNAs) are almost invisible to these old eyes. Repression
and epigenetic modifications are also handled well, and
are well-illustrated, except for Figure 5–23, 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 process-
ing of pre-mRNA, including splicing and poly(A) addi-
tion. This is a very fast-moving field and even with many
references to work published in 2010 and 2011, some
conclusions will have to be modified. 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 Drosoph-
ila has 95 exons and can be spliced differentially to yield
more than 30,000 different proteins from the same pre-
mRNA 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 RNA-
binding proteins called Nova are responsible for cell-
specific splicing in the brain, resulting in specific proteins
found in synapses? The latter work is from the lab of
Robert Darnell, the author’s son, who clearly followed the
advice Jim offered to anyone who listened: study neuro-
biology.
The final part of this large chapter discusses small
RNAs, returning briefly to the historic approach by de-
scribing 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 Ruvkun’s finding 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 nema-
todes and observed significant 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 floodgates 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 per-
oration: “Those of us among the thousands of us who did
not discover the structure of DNA, the universal genetic
code, or the first proteins that can and do control genes
can still justifiably enjoy our achievements and be secure
in the knowledge that we took part in a great and
continuing enterprise.” Amen.
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