RNA structure and evolution

2013 19: vii-xiRNA
R.R. Gutell
2012)−Woese (1928
You tell Carl that some of my best friends are Eukaryotes: Carl R.
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You tell Carl that some of my best friends are Eukaryotes:
Carl R. Woese (1928–2012)
I cherished the opportunity to have Dr. Carl Woese as one of
my mentors and collaborators. While I and many others are
deeply saddened by his death on December 30, 2012, we are
so grateful for his many contributions to science and for the
many ways he enlightened our lives. His wisdom and encour-
agement are most appreciated.
Shortly after I started graduate school in the late 1970s to
study the structure and function of the ribosome at the
University of California at Santa Cruz, my advisor, Dr. Harry
Noller, posted this November 3, 1977, New York Times article
on the front door to his lab: “Scientists discover a form of life
that predates higher organisms” (Woese and Fox 1977a).
LittledidIknowatthetimethatthisdescriptionaboutthethird
form of life, discovered by Drs. Carl Woese and George Fox,
would establish the foundation for my entire professional ca-
reer, influence the scientific careers for many (many) scien-
tists, while making a profound contribution to several
primary disciplines in biology.
One of my first projects in Harry’s lab was to help solve
the secondary structure for the Escherichia coli 16S and 23S
rRNA. We quickly determined that the number of possible
rRNA secondary structure models was more than the num-
ber of elemental particles in the universe. This problem was
exacerbated by the lack of adequate energy values for all of
the simple RNA structural elements and motifs that collec-
tively form the higher-order structure for RNA molecules.
Thus we could not discern, based on first principles, which
of the many possible structure models were correct. I consid-
ered my first project in Harry’s lab to be a failure. Fortunately
Harry and Carl were good friends and Carl encouraged Harry
to use comparative methods to identify the 16S rRNA sec-
ondary structure that is common for a set of 16S rRNA se-
quences from different organisms. I have fond memories of
Carl visiting Santa Cruz to collaborate on this project. The
minimal secondary structure for the 16S rRNA (and later
23S rRNA) was determined in the early 1980s (Woese et al.
1980; Noller et al. 1981). Although people questioned at
the onset this nonbiophysical method of determining an
RNA’s secondary structure, scientists started to embrace this
method when the resulting secondary structure models were
consistent with their experimental results while providing a
better framework to interpret their experiments and speculate
about the structure and function of their biological system.
These 16S and 23S rRNA secondary structure models were
refined as the number and diversity of sequences increased
dramatically in parallel with the development of more com-
putationally rigorous covariation methods. The authenticity
of these comparative structure models was evaluated when
the high-resolution crystalstructuresfor the 30S and 50S ribo-
somal subunits were determined in 2000. I doubt that Gregor
Mendel could have done better. Approximately 98% of the
base pairs predicted with the covariation methods were pre-
sent in these high-resolution crystal structures (Gutell et al.
2002).
Once it became apparent to a growing number of molec-
ular biologists that comparative methods had the potential
to accurately determine an RNA’s secondary structure, some
people, including me, believed that comparative methods
“had served its purpose.” It was now time for other methods
to elucidate more details about RNA structure. Not according
to Carl and Harry. I received a paper from Carl, written by
Michael Levitt (1969), revealing that comparative methods
were used to accurately predict a few of the tertiary structure
interactions in tRNA. Thus I turned down a few postdoc
offers in experimental labs to continue my comparative anal-
ysis of the rRNAs in Carl’s lab at the University of Illinois. We
(Photo Courtesy Don Hamerman)
RNA (2013) Published by Cold Spring Harbor Laboratory Press. Copyright © 2013 RNA Society. vii

identified several tertiary structure base pairs, some com-
posed of canonical G:C, A:U, and G:U base pairs, others com-
posed of noncanonical base pair and exchanges, including
U:U <-> C:C, G:A <-> A:G, A:A <-> G:G, G:U < > A:C.
Other irregular structural elements were identified, including
single or lone base pairs, lone base pairs capped with a 3-nu-
cleotide hairpin loop, base pairs forming pseudoknots, paral-
lel (vs. anti-parallel) arrangement of consecutive base pairs,
base triples, and other novel structural elements (Gutell
et al. 1986; Woese and Gutell 1989; Gutell and Woese 1990).
All of these noncanonical structural elements were present
in the high-resolution crystal structure (Gutell et al. 2002).
These latter studies revealed that comparative analysis can
not only identify the correct canonical secondary structure,
but can begin to identify and characterize new types of struc-
tural elements.
After these discoveries of irregular structural elements
based on covariation analysis, some of us questioned again
whether comparative analysis can reveal more about RNA
structure. And again Carl had more to contribute. Carl had
an amazing ability to see patterns in the secondary structure
diagrams. While in hindsight it is obvious, Carl first noticed
that the majority of the hairpin loops in 16S rRNA had only
four nucleotides, and only 10 or so of the 256 sequences of
length four were present at nearly all of the bacterial tetra-
loops (Woese et al. 1990). We observed other biased distribu-
tions of nucleotides on several structural elements, including
the large abundance of unpaired adenosines (Gutell et al.
1985, 1994). Thus, comparative analysis could be used to
identify and characterize RNA structural motifs, the basic
building blocks of RNA structure.
Possibly one of the most audacious statements Carl wrote
(from my perspective) was published in 1983. At that time,
Carl and Harry were publishing our first “minimal” compar-
ative secondary structure models for the 16S and 23S rRNAs.
While some people were skeptical of these models, both of my
mentors had utmost confidence in them. Carl knew that com-
parative analysis could reveal more than “just” the secondary
structure base pairs:
“The comparative approach indicates far more than the
mere existence of a secondary structural element; it ulti-
mately providesthe detailed rules for constructing the func-
tional form of each helix. Such rules are a transformation
of the detailed physical relationships of a helix and perhaps
even reflection of its detailed energetics as well. (One might
envision a future time when comparative sequencing pro-
vides energetic measurements too subtle for physical chem-
ical measurements to determine.)” (Woese et al. 1983.)
My lab and others have used comparative methods to derive
pseudo-energies (statistical potentials) that are a bit more
accurate than experimentally determined energy values for
structural elements (Do et al. 2006; Andronescu et al. 2010;
Gardner et al. 2011).
Carl did not mince his words when he believed that the
scientists were working under the pretenses of a faulty para-
digm. Possibly a better example of Carl’s chutzpah follows in
his presentation entitled “Just so stories and Rube Goldberg
machines: Speculations on the origin of the protein synthetic
machinery” at the 1980 ribosome conference (Woese 1980).
Carl wrote:
“The organizers of this Symposium have asked me to
speak on the topic ‘Speculations on the Origin of the
Protein Synthetic Machinery’, which I have appropriately
retitled ‘Just-So Stories, and Rube Goldberg Machines’.
The topic is a challenging but frustrating one. It is chal-
lenging because in order to address it properly one is forced
into the much-needed reexamination of our concept of
translation and its relationship to Biology as a whole. It
is frustrating for two reasons: For one, unavoidably I
will have to present a Just-So Story. What do we really
know about how translation works at the molecular level?
We know nothing! How then, does one explain the evolu-
tion of an unknown mechanism? By a Just-So Story! My
second reason is that this presentation will at best elicit a
ho-hum response; the field is atune to a paradigm that
sees little value in understanding how translation evolved.
From the set of codon assignments on down all facets of the
translation mechanism are taken as arising by ‘historical
accident’, as being un-repeatable evolutionary events.
This can and has given rise to the prejudice that the trans-
lation apparatus is basically a Rube Goldberg Machine—
some incongruous assemblage of parts, where knowing
even ninety percent of the mechanism would not permit
one to predict the remaining ten percent. The Rube
Goldberg view not only generates disinterest in the mech-
anism’s evolution, but also leads to a feeling that there is
no point in attempting to think, to theorize about a mech-
anism that is unknowable a priori; one’s approach needs
to be ‘strictly empirical’. If this presentation serves no other
purpose, I should like it to raise the issue of the design of
the translation apparatus; Is it really a Rube Goldberg
Machine? Is there a simple mechanism at its core? By
what principles does it achieve its low noise level? Does
it possess an understandable evolutionary structure?”
Although these accomplishments are most significant,
Carl is mostly recognized for his studies on the evolution of
organisms, not RNA.
The development of Carl’s scientific inquiries was influ-
enced by the discovery of DNA’s double helix structure
near the time he completed his PhD at Yale (Woese 2004).
Like others at that time, he was intrigued with the genetic
code. However, the simple assignments of codons to amino
acids did not satisfy his curiosity about translation. Carl start-
ed questioning what was special about the relationship be-
tween the genotype and phenotype, the mechanism of
translation, and how these relationships and mechanisms
evolved. And to resolve these issues, Carl realized that a
viii
In memoriam

“universal phylogenetic framework” was needed. Carl also
realized that the evolutionary relationships between bacteria
were unknown, although their abundance was significantly
greater than that for animals and plants. The seeds for at least
part of Carl’s scientific career were planted.
Carl (and George Fox) also realized that any attempt to
understand the evolution of one of the most fundamental
mechanisms of the cell—protein synthesis—brought them
face to face with the origin of cellular life. Thus, not only
were they trying to reconstruct the phylogenetic relationships
for all organisms, they were trying to decipher the molecular
and cellular events that occurred shortly before and after the
origin of cellular life. They published one of my favorite pa-
pers, “The Concept of Cellular Evolution” (Woese and Fox
1977b), the year I started graduate school. The concept of
the Progenote—the predecessor to the cellular life as we cur-
rently know it—was introduced.
Utilizing new nucleic-acid sequencing technology devel-
oped by Fred Sanger, Carl started his majestic effort to deter-
mine the evolutionary relationships for prokaryotes and relate
them with eukaryotes. Carl and George reasoned (Woese and
Fox 1977a; Woese 1987) that the analysis of “comparable
structures” that are (1) present in all life forms and (2) at
the core of the fundamental cellular mechanisms in the cell
is necessary to reconstruct phylogenetic relationships that
span the entire tree of life. That core, as they defined it, was
the ribosome’s translation of the cell’s genotype to its pheno-
type. They rationalized that the evolution of the ribosomal
RNAs, to a first approximation, would be neutral to the envi-
ronment and their evolution would be slower than other
genetic sequences. Although the ribosomal RNA satisfied
these criteria, the majority of macromolecular sequences
evolves too quickly and thus can only be used to determine
phylogenetic relationships for a small region of the phyloge-
netic tree, not for the full spectrum of life forms. Since it
was also rationalized that RNA was present before DNA and
proteins (Woese 1967; Crick 1968; Orgel 1968) and that ribo-
somal RNA was directly associated with the translation of the
cell’s genotype to its phenotype (Woese and Fox 1977b), an
analysis of rRNA might reveal the early stages in the origin
of life during the transition between the progenote and
the original forms of the Archaea, Bacteria, and Eukaryotes.
These seminal concepts were the foundation for the determi-
nation of rRNA sequences for organisms that span the entire
tree of life, resulting in (1) the discovery of the Archaea as the
third form of life; (2) the first phylogenetic trees that contain
representative organisms from the full spectrum of all liv-
ing forms (Woese 1987, 2000); (3) the massive community
effort to determine rRNA sequences from all forms of life re-
sulting in the largest collection of sequence data for any one
gene; (4) the use of rRNA sequences for medical diagnostic
purposes (e.g., Gen-Probe, http://www.gen-probe.com/
science/#technologies-3); and (5) the analysisof microbiomes
with16SrRNAsequencingrevealinghowprevalent,pervasive,
and important Bacteria and Archaea are for the survival and
health of multicellular organisms and different environments
on earth. One editorial published in Nature Reviews
Microbiology (Editorial 2011) described a compelling reason
why Carl Woese should win the Nobel Prize:
“Carl Woese has completely changed the way we view the
relationships between all organisms on Earth, revealed the
presence of a previously unrecognized domain and provid-
ed us with a tool that has begun to elucidate the complex
composition of the human microbiome, which constitutes
90% of the genetic diversity of our bodies and has been
called the second human genome. It is difficult to think
of more-fundamental discoveries that are affecting the
way we think about the environment and human health
alike. As the attentions of the scientific community turn
once again to the decisions of the Nobel committee, per-
haps it is time to campaign for Carl Woese to receive the
recognition that he deserves.”
Carl also addressed a multitude of related topics, including
mitochondrial origins (Woese 1977; Yang et al. 1985); the ge-
netic code (Woese 1965a,b, 1967, 1969; 1970a,c; 1973; Woese
et al. 1966); speculated that RNA came before DNA and pro-
teins, which published a year before Francis Crick and Leslie
Orgel published similar speculations (Woese 1967; Crick
1968; Orgel 1968; Orgel and Crick 1993); more speculations
about the mechanisms of translation (Woese 1970b; Woese
2001); and the use of comparative analysis to predict the (cor-
rect) secondary structure for 5S rRNA (Fox and Woese 1975).
“A new biology for a new century” is one of my favorite
articles written by Carl (Woese 2004). Freeman Dyson de-
scribes it with much eloquence (Dyson 2007):
“Whatever Carl Woese writes, even in a speculative vein,
needs to be taken seriously. In his ‘New Biology’ article, he
is postulating a golden age of pre-Darwinian life, when
horizontal gene transfer was universal and separate species
did not yet exist. Life was then a community of cells of var-
ious kinds, sharing their genetic information so that clever
chemical tricks and catalytic processes invented by one
creature could be inherited by all of them. Evolution was
a communal affair, the whole community advancing in
metabolic and reproductive efficiency as the genes of the
most efficient cells were shared. Evolution could be rapid,
as new chemical devices could be evolved simultaneously
by cells of different kinds working in parallel and then re-
assembled in a single cell by horizontal gene transfer.
“But then, one evil day, a cell resembling a primitive
bacterium happened to find itself one jump ahead of its
neighbors in efficiency. That cell, separated itself from
the community and refused to share. Its offspring became
the first species of bacteria—and the first species of any
kind—reserving their intellectual property for their own
private use. With their superior efficiency, the bacteria
continued to prosper and to evolve separately, while the
rest of the community continued its communal life.
www.rnajournal.org ix
In memoriam

Some millions of years later, another cell separated itself
from the community and became the ancestor of the
archea. Some time after that, a third cell separated itself
and became the ancestor of the eukaryotes. And so it
went on, until nothing was left of the community and
all life was divided into species. The Darwinian interlude
had begun.”
Now quoting directly from Carl’s “A new biology for a new
century” (Woese 2004):
“Let’s stop looking at the organism purely as a molecular
machine. The machine metaphor certainly provides in-
sights, but these come at the price of overlooking much
of what biology is. Machines are not made of parts that
continually turn over, renew. The organism is. Machines
are stable and accurate because they are designed and built
to be so. The stability of an organism lies in resilience, the
homeostatic capacity to reestablish itself. While a machine
is a mere collection of parts, some sort of ‘sense of the whole’
inheresintheorganism,aqualitythatbecomesparticularly
apparent in phenomena such as regeneration in amphibi-
ans and certain invertebrates and in the homeorhesis ex-
hibited by developing embryos.
“If they are not machines, then what are organisms? A
metaphorfarmoretomylikingisthis.Imagineachildplay-
ing inawoodland stream, poking a stick into an eddy in the
flowing current, thereby disrupting it. But the eddy quickly
reforms. The child disperses it again. Again it reforms, and
the fascinating game goes on. Thereyou have it! Organisms
are resilient patterns in a turbulent flow—patterns in an
energy flow. A simple flow metaphor, of course, fails to cap-
ture much of what the organism is. None of our representa-
tions of organism capture it in its entirety. But the flow
metaphor does begin to show us the organism’s (and biol-
ogy’s) essence. And it is becoming increasingly clear that
to understand living systems in any deep sense, we must
come to see them not materialistically, as machines, but
as (stable) complex, dynamic organization.
“Twenty-first century biology will concern itself with
the great ‘nonreductionist’ 19th
century biological prob-
lems that molecular biology left untouched. All of these
problems are different aspects of one of the great problems
in all of science, namely, the nature of (complex) organiza-
tion. Evolution represents its dynamic, generative aspect;
morphology and morphogenesis represent its emergent,
material aspect. One can already see the problem of the
evolution of cellular organization coming to the fore. And
because of both its pressing practical and its fundamental
nature, the problem of the basic structure of the biosphere
is doing so as well.
“My own career is one of the links between biology’s
reductionist molecular past and its holistic future.”
Although Carl had already discovered the third domain of
life, proposed that RNA came before DNA and proteins,
wrote eloquently and forcibly about the genetic code and
translation prior to the time I met him, he was not (at that
time) a member of the National Academy of Sciences. And I
sensed that Carl was disappointed, for good reason, that his
many contributions to science were not properly recognized.
I fondly remember visiting Harry in Santa Cruz a few months
after I started my postdoc with Carl. I told Harry that Carl said
that if he does not get elected into the National Academy
within the next two years, he will reject it in the event he is
elected into this prestigious academy. Harry started laughing
and laughing (and laughing). I asked, “What is so funny?”
“Carl made the same statement to me three years ago.” Carl
was elected into the National Academy two years later. Carl
has received many awards, most of which were received after
becoming a member of the National Academy. Carl was a
MacArthur Fellow in 1984, was made a member of the
National Academy of Sciences in 1988, received the Leeu-
wenhoek Medal (microbiology’s highest honor) in 1992 and
the Selman A. Waksman Award in Microbiology in 1995
from the National Academy of Sciences, and was a National
Medal of Science recipient in 2000. In 2003, he received the
Crafoord Prize from the Royal Swedish Academy of Sciences
“for his discovery of a third domain of life.” In 2006, he was
made a foreign member of the Royal Society. I sensed that
Carl was indeed proud of his recognition, but more proud of
his contributions to science.
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R.R. Gutell
Section of Integrative Biology
University of Texas at Austin
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