Nucleic acidsNucleic acid structure — 50 not out!Editorial overviewDavid MJ Lilley and Carl C Correll

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Current Opinion in Structural Biology 2003,

13:263–265

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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: d.m.j.lilley@dundee.ac.uk

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: ccorrell@uchicago.edu

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

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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

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