Nucleic acidsNucleic acid structure — 50 not out!Editorial overviewDavid MJ Lilley and Carl C Correll
263
Current Opinion in Structural Biology 2003,
13:263–265
0959-440X/03/$ – see front matter
� 2003 Elsevier Science Ltd. All rights reserved.
DOI 10.1016/S0959-440X(03)00068-X
David MJ Lilley
Division of Biological Chemistry and Molecular
Microbiology, School of Life Sciences,University of Dundee, Dow Street,
Dundee DD1 5EH, UK
e-mail: [email protected]
David Lilley has a long-standing
interest in helical branchpoints in DNA
and RNA. He solved the structure of
the Holliday junction in DNA and has
made extensive studies of the
interaction with resolving enzymes. He
is interested in folding and catalysis in
RNA, and has studied thehammerhead, hairpin and VS
ribozymes.
Carl C Correll
Department of Biochemistry and Molecular
Biology, University of Chicago, Chicago,
IL 60637, USA
e-mail: [email protected]
The Correll laboratory studies the
structural underpinnings of essentialRNA–protein interactions. One area of
study addresses how translation
factors and toxins recognize a
universal RNA structure in ribosomes:
the sarcin/ricin loop. Another area
focuses on the assembly of the U3
ribonucleoprotein complex and its role
in ribosome biogenesis.
On the 50th anniversary of the discovery of the double-helical structure of
DNA, considerable emphasis has been placed on history. Despite that,
nucleic acid structure remains a vibrant area, with biological consequences at
many levels. And, although few would probably dispute this for RNA, DNA
is still capable of springing structural surprises, as we shall see.
Right from the inception, structural studies of nucleic acids have often
benefited from the application of new approaches and this remains true
today. Single-molecule experiments are now having enormous impact on
studies of both DNA and RNA. These take a variety of forms, but all
transform our analysis of the dynamic properties of nucleic acids, giv-
ing a radically fresh perspective on many processes. The group of David
Bensimon has pioneered the technique of simultaneously stretching and
twisting DNA using magnetic beads while measuring the applied force. This
provides completely new information on DNA structure under extreme
torsional stresses, to say nothing of a very direct way to introduce positive or
negative superhelical stress into DNA. In addition, these experiments now
provide a new and powerful way of doing enzymology on DNA, with
topoisomerases and polymerases for example. The applications of single-
molecule force experiments and single-molecule spectroscopy have prob-
ably only just scratched the surface of what is possible and tremendously
exciting times lie ahead for sure.
One of the more structurally adventurous forms of DNA is adopted by
guanine-rich sequences of the kind found at the telomeres of chromosomes.
These are four-stranded helices, based on the formation of tetrads (also
known as quartets) of guanine bases. It had been found that, whereas four
separate strands tended to associate in a parallel manner, an intermolecular
structure formed from a single strand folding back on itself could adopt a
variety of antiparallel structures. Because the telomere presents a guanine-
rich single-stranded 30 overhang, it was assumed that, if a tetraplex formed, it
would be of the antiparallel kind. However, DNA has caught us out again.
Stephen Neidle and Gary Parkinson have presented a remarkable new
crystal structure in which a folded-back DNA does a kind of molecular loop-
the-loop, so that the local geometry of the tetraplex is in fact parallel. The
biological ramifications have not yet been worked out for this structure, but
it is a very stimulating development.
Sequence-specific recognition of DNA lies at the heart of gene regulation
and development. Proteins have had billions of years of evolution to perfect
this art. Chemists have had rather less time to try to emulate the specificity
that Nature achieves, but recent results have been extraordinary. Peter
Dervan and Benjamin Edelson have summarised the work of Dervan’s
www.current-opinion.com Current Opinion in Structural Biology 2003, 13:263–265
group to develop polyamide compounds that can be
designed to bind to specific sequences with high affinity
and used to regulate gene expression in cells. These
compounds are distantly related to natural species such
as the distamycins and bind in the minor groove of DNA.
This in itself is remarkable, as the minor groove is very
much less promising than the major groove for making
specific contacts; most specific DNA–protein contacts are
made in the major groove. Yet, because of their clever
design, Dervan’s compounds evidently recognise subtle
aspects in the relatively featureless minor groove.
For a long time, the only atomic-level structural informa-
tion on RNA came from crystallography of tRNA. But, in
the past few years, this situation has changed out of all
recognition and the determination of high-resolution
structures of both ribosomal subunits has provided so
much new information that the problem is now one of
classification. Eric Westhof has turned his customary
insight to this problem, beginning with a valuable way
of summarising base interactions in a simple and clear
manner. Neocles Leontis and Westhof have summarised
the various motifs that are now accepted as commonly
occurring elements in RNA, including a variety of stem-
loops, A-minor interactions (a generalisation of a number
of previously identified structural features), kink-turns
and more.
Folding and function of RNA are completely interwoven.
And just as there is the famous ‘protein folding problem’,
so there is a similar challenge to understand how RNA
goes from a linear sequence of nucleotides to a three-
dimensional folded functional molecule. Of course, RNA
is an utterly different polymer from protein. Its charged
phosphodiester backbone places it in a world dominated
by electrostatics, where interactions with metal ions
become critical. These comparisons are well made by
Tobin Sosnick and Tao Pan, who provide a refreshing
perspective on the whole problem of RNA folding, both
kinetic and thermodynamic.
Some of the most interesting RNA species exhibit cat-
alytic activity, which throws up a new set of questions.
The origins of catalytic activity have not been fully
established for any ribozyme to date and most aspects
of catalysis are controversial. The final three reviews
address different aspects of RNA catalysis.
Metal ions are required for RNA folding and correct
folding is necessary to provide the environment in which
catalysis can occur. Bound metal ions can also play a direct
role in RNA-mediated reactions as well, in general acid-
base catalysis, as Lewis acids, or in electrophilic catalysis.
The experimental difficulty often lies in distinguishing
the specific ion effects among the myriad of nonspecific
interactions with the RNA polyelectrolyte. This problem
is addressed by Victoria DeRose, who discusses the
complexities of analysing phosphorothioate substitution
and rescue experiments for the hammerhead and group I
ribozymes. She also discusses the application of modern
electron spin resonance experiments to the analysis of the
metal ion environment in RNA molecules.
RNase P is a ubiquitous ribonucleoprotein complex
required to process the 50 terminus of pre-tRNA.
Michael Harris and Eric Christian discuss the bacterial
RNase P, in which the reaction is brought about by the
400-nucleotide RNA component. Uniquely, this ribo-
zyme carries out hydrolysis of the phosphodiester bond,
unlike the transesterification reactions of the nucleolytic
ribozymes, as exemplified by the hammerhead ribo-
zyme. The RNA can be divided into catalytic and
specificity domains — the structure of the latter has just
been solved by X-ray crystallography. Once again, metal
ions appear to play a key role in the reaction catalysed by
RNase P.
Finally, we turn to the biggest ribozyme of them all,
which catalyses what is arguably the most important
reaction in the cell — peptidyl transferase. Although
long suspected, the solution of the crystal structure of
the 50S ribosomal subunit by Steitz and Moore showed
that the peptidyl transferase centre is devoid of protein
and that the ribosome is therefore a ribozyme. It can thus
be speculated that the ribosome is a kind of three-
billion-year-old molecular fossil of an earlier RNA world.
The peptidyl transferase reaction is very different from
those catalysed by other natural ribozymes, which all
carry out variations on a theme of phosphoryl transfer
reactions; by contrast, condensation of amino acids
requires the attack of the a-amino group on a carbonyl
centre (i.e. an sp2-hybridised carbon atom) to generate a
tetrahedral intermediate, which must then be resolved
in a second step. The crystal structure of the 50S subunit
with a bound tetrahedral intermediate analogue sug-
gested that general base catalysis by an adenine nucleo-
base might be a significant component of the catalytic
mechanism. However, this was almost immediately con-
tradicted by data from mutants in vivo and in vitro.
Marina Rodnina and Wolfgang Wintermeyer set out to
determine if the contribution of the nucleobase to cat-
alysis could be masked by conformational changes that
are thought to limit the overall rate of protein synthesis.
Detailed analyses of the pH dependence of the peptidyl
transferase reaction under conditions in which the chem-
istry is expected to be rate limiting revealed that an RNA
nucleobase with a pKa close to neutrality contributes to
catalysis. Mutation of a suspected adenine (A2451U)
leads to a loss of pH dependence and the residual
catalysis is probably due to substrate positioning. In
fact, it is probably generally true that RNA catalysis is
a multifactorial process, which is why it has been so
difficult to sort out in most cases. Interestingly, despite
the differences in chemistry, the factors leading to
264 Nucleic acids
Current Opinion in Structural Biology 2003, 13:263–265 www.current-opinion.com
catalytic rate enhancement are very similar to those
operating in other ribozymes, perhaps showing that
RNA must exploit its relatively limited resources to
the maximum.
We thank all the authors for a uniformly high standard
of reviews. They certainly demonstrate beyond doubt
that nucleic acid structural studies are as exciting as
ever half a century on from the elucidation of the
double helix.
David MJ Lilley
Carl C Correll
May 2003
Editorial overview Lilley and Correll 265
www.current-opinion.com Current Opinion in Structural Biology 2003, 13:263–265