Table Of ContentPreface
The importance of the role RNA plays in all aspects of gene expression has been understood by
molecular biologists and biochemists since the late 1950s. Nevertheless, relative to what was going
on in the DNA and protein fields, RNA biochemistry remained a backwater for many years primarily
because RNA is hard to work with. For example, unless handled carefully, RNAs are rather prone to
hydrolytic degradation, and most of the RNAs abundant in nature have molecular weights so large that
for a long time it seemed unlikely that anything useful could be learned about them using the physical
and chemical techniques of the day. In addition, many biologically important RNAs are so rare that it is
difficult to prepare enough of any one of them from natural sources to do all the experiments one would
like. Finally, for many years, by comparison with the protein world, the RNA universe appeared to he
very small, consisting only of transfer RNAs, ribosomal RNAs, messenger RNAs, and a few viral RNAs.
Why spend one's career struggling to understand the properties of a class of macromolecules so difficult
and so limited?
The mind set of those in the RNA field has slowly been transformed from a somewhat pessimistic
resignation to near manic optimism by the events of the last twenty years. Powerful methods have been
developed for sequencing RNA, and a rich variety of chemical and genetic methods is now available
for determining the functional significance of single residues in large RNAs, and even that of individual
groups within single residues. On top of that, the supply problem has been solved. Chemical and
enzymatic methods now exist that make it possible to synthesize RNAs of any sequence in amounts
adequate for even the most material-hungry experimental techniques. In many other respects, RNA is
easier to work with today than protein. In addition, the RNA universe has expanded. Scores of new
RNAs have been discovered, most of them in eukaryotic organisms, that perform functions of which
the biochemical community was entirely ignorant in the 1960s, when the first blossoming of the RNA
field occurred. Additional stimulus was provided in the 1980s by the discovery that two different
RNAs possess catalytic activity, and several additional catalytic RNAs have since been identified. Their
existence has led to renewed interest in the possibility that the first organisms might have used RNA both
as genetic material and as catalysts for the reactions required for their survival. Francis Crick's reflection
(in 1966) on an RNA molecule's versatility ("It almost appears as if tRNA were Nature's attempt to
make an RNA molecule play the role of a protein") can now be extended to many RNA species. One
interesting offshoot of these developments has been the invention of a new field of chemistry that is
devoted to the production of synthetic RNAs that have novel ligand binding and catalytic activities.
Finally, belatedly NMR spectroscopists and X-ray crystallographers have begun solving RNA structures.
This volume covers the full range of problems being addressed by workers in the RNA field today.
Fach chapter has been contributed by a scientist expert in the area it covers, and is thus a reliable guide
for those interested in entering the field. The Editors hope that those patient enough to read the entire
book will come away with an appreciation of the rapid progress now being made in the RNA field, and
will sense the excitement that now pervades it. RNA biochemistry is destined to catch up with DNA and
protein biochemistry in the next 10 or 15 years, and it is certain that important new biological insights
will emerge in the process.
DIETER SOLE
Editor
Contributors
Dr. S. Altman
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06511,
USA
Dr. B.L. Bass
Howard Hughes Medical Institute, University of Utah, 6110a Eccles Institute of Human Genetics,
Building 533, Salt Lake City, UT 84112, USA
Dr. J.H. Gate
Departments of Chemistry and Molecular Cell Biology, Sinsheimer Laboratories, University of Califor
nia, Berkeley, CA 95064, USA
Dr. D.M. Crothers
Department of Chemistry, Yale University, New Haven, CT 06520, USA
Dr. A.E. Dahlberg
Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Box G-J4, Provi
dence, RI02912, USA
Dr. J.A. Doudna
Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven,
CT 06520-8114, USA
Dr. F. Eckstein
Abteilung Chemie, Max-Planck-Institut fUr Experimentelle Medizin, Hermann-Rein-Strasse 3, D-37075
Gottingen, Germany
Dr. A.D. Ellington
Department of Chemistry, Institute for Cellular and Molecular Biology, University of Texas at Austin,
26th and Speedway, Austin, TX 78712, USA
Dr. C. Florentz
UPR 9002 du CNRS, Institut de Biologic Moleculaire et Cellulaire, 15, rue Rene Descartes, F-67084
Strasbourg-Ceex, France
Dr. M.A. Garcia-Blanco
Departments of Genetics, Microbiology and Medicine, Duke University Medical Center, Durham, NC
27710, USA
Dr. S. Ghosh
Department of Genetics, Duke University Medical Center, Durham, NC 27710, USA
Dr. R. Giege
UPR 9002 du CNRS, Institut de Biologic Moleculaire et Cellulaire, 15, rue Rene Descartes, F-67084
Strasbourg-Cedex, France
X Contributors
Dr. S.T. Gregory
Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Box G-J4, Provi
dence, RI02912, USA
Dr. M. Helm
UPR 9002 du CNRS, Institut de Biologic Moleculaire et Cellulaire, 15, rue Rene Descartes, F-67084
Strasbourg-Cedex, France
Dr. H. Kawasaki
National Institute for Advanced Interdisciplinary Research, AIST, MITI, Tsukuba Science City 305-8562,
Japan
Dr. Y. Komatsu
Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan
Dr. T. Kuwabara
Institute of Applied Biochemistry, University of Tsukuba, Tennoudai 1-1-1, Tsukuba Science City 305-
8572, Japan
Dr. L.A. Lindsey-Boltz
Program in Molecular Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA
Dr. D.H. Mathews
Department of Chemistry, University of Rochester, Rochester, NY 14627-0216, USA
Dr. J.A. McCloskey
Department of Medicinal Chemistry, University of Utah, 30 S. 2000 East, Salt Lake City, UT 84112-5820,
USA
Dr. RB. Moore
Departments of Chemistry and Molecular Biophysics and Biochemistry, Yale University, New Haven, CT
06520-8017, USA
Dr. M. O'Connor
Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Box G-J4, Provi
dence, RI02912, USA
Dr. J. Ohkawa
National Institute for Advanced Interdisciplinary Research, AIST, MITI, Tsukuba Science City 305-8562,
Japan
Dr. M. Ohman
Department of Molecular Genome Research, Stockholm University, SE-10691 Stockholm, Sweden
Professor E. Ohtsuka
Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan
Dr. R. Parker
Departments of Molecular and Cellular Biology & Biochemistry and Howard Hughes Medical Institute,
University of Arizona, Tucson, AZ 85721, USA
Dr. M.R Robertson
Department of Chemistry, Institute for Cellular and Molecular Biology, University of Texas at Austin,
26th and Speedway, Austin, TX 78712, USA
Dr. J.K. Strauss-Soukup
Department of Molecular Biophysics and Biochemistry, Yale University, 260 Whitney Avenue, New Haven,
CT 06520, USA
Current address: Chemistry Department, Creighton University, 2500 California Plaza, Omaha, NE
68178, USA
Contributors xi
Dr. S.A. Strobel
Department of Molecular Biophysics and Biochemistry, Yale University, 260 Whitney Avenue, New Haven,
CT 06520, USA
Professor R.H. Symons
Department of Plant Science, Waite Campus, The University of Adelaide, Glen Osmond, SA 5064,
Australia
Dr. K. Taira
Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo,
Hongo, Tokyo 113-8656, Japan
Dr. S. Tharun
Departments of Molecular and Cellular Biology & Biochemistry and Howard Hughes Medical Institute,
University of Arizona, Tucson, AZ 85721, USA
Dr. D.H. Turner
Department of Chemistry, University of Rochester, Hutchinson B08, Rochester, NY 14627, USA
Dr. N.K. Vaish
Ribozyme Pharmaceuticals, Inc., Boulder, CO 80301, USA
Dr. S. Verma
Department of Chemistry, Indian Institute of Technology, Kanpur 208016 (UP), India
Dr. A. Vioque
Instituto de Bioquimica Vegetal y Fotosintesis, Universidad de Sevilla - CSIC, Americo Vespucio s/n
41092 Sevilla, Spain
Dr. M. Warashina
Institute of Applied Biochemistry, University ofTsukuba, Tennoudai 1-1-1, Tsukuba Science City 305-
8572, Japan
Dr. T. Xia
Department of Chemistry, University of Rochester, Rochester, NY 14627-0216, USA
1
A Spectroscopist's View of
RNA Conformation: RNA Structural
Motifs
PETER B. MOORE
Yale University, New Haven, CT, USA
1.1 INTRODUCTION 2
1.2 THE DETERMINATION OF RNA STRUCTURES BY NMR 2
1.2.1 NMR Fundamentals 2
7.2.2 Chemical Shift 3
1.2.3 Couplings and Torsion Angles 3
1.2.4 Spin-Lattice Relaxation: Nuclear Overhauser Effects and Distances 4
1.2.5 Spin-Spin Relaxation: Molecular Weight Limitations 5
1.2.6 Samples 6
1.2.7 Multidimensional NMR 6
7.2.5 Assignments 6
7.2.9 Helices and Torsion Angles 7
1.2.10 Distance Estimation 8
7.2.77 Structure Calculations 9
1.3 SOLUTION STRUCTURES AND CRYSTAL STRUCTURES COMPARED 9
1.3.1 On the Properties of Crystallographic Structures 9
1.3.2 Solution Structures 10
1.3.3 Constraints and Computations 10
1.3.4 Experimental Comparisons of Solution and Crystal Structures 11
7.5.5 New Approaches 12
1.4 LESSONS LEARNED ABOUT MOTIFS BY NMR 12
1.4.1 RNA Organization in General 13
1.4.2 Terminal Loops 13
7.4.2.7 U-turns 13
7.4.2.2 Tetraloops 14
1.4.2.3 Other terminal loops 15
1.4.3 Internal Loops 15
1.4.3.1 Symmetric internal loop motifs 15
1.4.3.2 Asymmetric internal loop motifs 16
1.4.4 Pseudoknots 17
1.5 REFERENCES 17
2 A Spectroscopist's View ofRNA Conformation: RNA Structural Motifs
1.1 INTRODUCTION
Biologically, RNA mediates between DNA and protein — DNA makes RNA makes protein — and
RNA is also intermediate between DNA and protein chemically. Some RNAs are carriers of genetic
information, like DNA, and others, e.g. transfer RNAs and ribosomal RNAs, are protein-like. Their
functions depend on their conformations as much as their sequences, and some even have enzymatic
activity.
Even though RNA biochemists have recognized their need for structures almost as long as protein
biochemists, far more is known about proteins than RNAs. Coordinates for over 8000 proteins have been
deposited in the Protein Data Bank, but the number of RNA entries is of the order of 100, and many of
them describe RNA fragments, not whole molecules.
All the RNA structures available before 1985 were crystal structures, and X-ray crystallography
remains the dominant method for determining RNA conformation. By the late 1980s, nuclear magnetic
resonance (NMR) had emerged as a viable alternative, but for many years, only a few structures a year
were being solved spectroscopically. In the last two years, the production rate has risen to roughly a
structure a month, and because the field is taking off, it is time RNA biochemists understand what the
structures spectroscopists provide are all about.
This chapter describes how RNA conformations are determined by NMR. The description provided is
intended to help biochemists understand what NMR structures are, not to teach them how to do it. The
chapter also summarizes what NMR has taught us about RNA motifs. For these purposes, a motif is any
assembly of nucleotides bigger than a base triple that has a distinctive conformation and is common in
RNAs.
1.2 THE DETERMINATION OF RNA STRUCTURES BY NMR
The behavior of all atoms that have non-zero nuclear spins can be studied by NMR, and the
predominant isotopes of two of the five elements abundant in RNA qualify in this regard: ^H and ^^R
Both have spins of V2. Those not content with the information ^H and ^^P spectra provide, can easily
prepare RNAs labeled with ^^C and/or ^^N, which are also spin-V2 nuclei (see below). Thus NMR
spectra can be obtained from all the atoms in a nucleic acid except its oxygens, for which no suitable
isotope exists. What can be learned from them?
The answer to this question, of course, can be mined out of the primary NMR literature, but it is
vast and much of it too technical for non-specialists. For that reason, rather than fill this chapter with
references its intended readers will find useless, I direct them here to a few secondary sources. For NMR
fundamentals, Slichter's book is excellent.^ It is complete, and its verbal descriptions are good enough so
that readers need not wade through its (many) derivations. Those interested in multidimensional NMR,
about which almost nothing is said below, can consult Goldman's short monograph,^ or the treatise of
Ernst and coworkers.^ Although a bit dated at this point, Wiithrich's book on the NMR of proteins and
nucleic acids is so useful the cover has fallen off the local copy."* A more technically oriented text on
protein NMR appeared recently, which is also useful.^
1.2.1 NMR Fundamentals
Nuclei that have spin (and not all do) have intrinsic magnetic moments, and thus orient like compass
needles when placed in magnetic fields. Because nuclei are very small, their response is quantized.
Spin-V2 nuclei orient themselves in magnetic fields in only two ways: parallel to it or antiparallel to
it. Because the energy associated with the parallel orientation is only slightly lower than that of the
antiparallel orientation, the number of nuclei in the parallel orientation is only slightly larger than the
number in the antiparallel orientation in any population of magnetically active atoms that has come to
equilibrium in a magnetic field. In the strongest available magnets, the excess is only a few per million.
The tiny bulk magnetization their collective alignment produces is what NMR spectroscopists study.
Sensitivity is not one of NMR's selling points!
An NMR spectrometer consists of a magnet to orient the nuclei in samples, a radio frequency
transmitter to perturb nuclear orientations in controlled ways, and a receiver to detect the electromagnetic
A Spectroscopisfs View ofRNA Conformation: RNA Structural Motifs 3
signals generated when the orientations of the magnetic moments of aUgned populations of nuclei
are perturbed. NMR spectrometers produce spectra, which are displays of the magnitude of these
electromagnetic signals as a function of perturbing frequency. A peak in such a display is a resonance.
1.2.2 Chemical Shift
Electromagnetic radiation causes the reorientation of spin-Vi nuclei that have become aligned in
external magnetic fields most efficiently when the product of Planck's constant and the frequency of
the reorienting radiation equals the difference in energy between their two possible orientations, i.e.
hv = AE. That frequency, the resonant frequency, is the one at which the intensity of a resonance in a
spectrum is maximum. The energy difference that determines a resonant frequency is the product of the
strength of the orienting magnetic field a nucleus experiences and its intrinsic magnetic moment.
The magnetic moments of all the nuclides relevant to biochemists were measured long ago, and they
differ so much that there is no possibility of confusing the resonances of one species with those of
another, a hydrogen resonance with a phosphorus resonance, for example. (N.B.: The resonant frequency
of protons in a 500 MHz NMR spectrometer is 500 MHz.)
The reason NMR interests chemists is that the magnetic field a nucleus experiences, and hence the
frequency at which it resonates, depends on its chemical context. Nuclei in molecules are surrounded by
electrons, which for these purposes are best thought of as particles in continual motion. When a charged
particle moves through a magnetic field, a circular component is added to its trajectory, and charged
particles moving in circles generate magnetic fields. Thus when a molecule is placed in a magnetic
field, it becomes a tiny solenoidal magnet the field of which (usually) opposes the external field. As
you would expect, both the direction and the strength of the field induced in a molecule this way
depend on its structure and on its orientation with respect to the inducing field. In solution, where rapid
molecular tumbling leads to averaging, orientation effects disappear, and the atom-to-atom variations
in the strength of the induced magnetic field within a molecule are reduced to a few millionths the
magnitude of the inducing field. Small though these induced field differences are, the contribution they
make to the total field experienced by each nucleus is easily detected because the receivers in modern
NMR spectrometers have frequency resolutions of about 1 part in 10^. Thus the proton spectrum of a
biological macromolecule is a set of resonances differing modestly in frequency, not a single, massive
resonance. Incidentally, all else being equal, the magnitude of each resonance produced by a sample is
proportional to the number of nuclei contributing to it.
The frequency differences that distinguish resonances in a spectrum are called chemical shifts, and
their importance cannot be overstated. Chemical shift differences are the primary way the resonances of
atoms at different positions in a population of identical molecules are distinguished from each other, and
if a spectrometer cannot resolve a large fraction of the resonances in an RNAs proton spectrum, little
progress can be made. (A resolved spectrum is one in which each resonance represents an atom in a
single position in the molecule of interest.)
Chemical shifts are usually reported using a relative scale the unit of which is the part per million
(ppm). The chemical shift of a resonances is 10^ times the difference between its resonant frequency and
the resonant frequency of an atom of the same type in some agreed-upon standard substance, divided by
the frequency of the standard resonance. A virtue of this scale is its independence of spectrometer field
strength. If a resonance has a chemical shift of 8 ppm in a 250 MHz spectrometer, its chemical shift
will be 8 ppm in a 800 MHz spectrometer also. By convention, if the frequency of a resonance is less
than that of the standard, its chemical shift is positive, and it is described as a down field resonance. Up
field resonances have negative chemical shifts. The proton spectrum of an RNA spans about 12 ppm, and
spectrometers can measure chemical shifts to about 0.01 ppm.
1.2.3 CoupUngs and Torsion Angles
The resonant behavior of atoms is also affected by its interactions with the magnetic fields generated
by all of the atomic nuclei in its neighborhood that have non-zero spins. In solution, most of these
interactions are averaged to zero by molecular tumbling, leaving the solution spectroscopist only a single
kind of internuclear interaction to worry about: J-coupling. If in a solution of identical molecules, a
4 A Spectroscopisf s View ofRNA Conformation: RNA Structural Motifs
spin-V2 atom at one position is /-coupled to a single spin-V2 atom at another, that atom will contribute
two closely spaced resonances to the molecule's spectrum, not the single resonance otherwise expected.
More complex splitting patterns arise when an atom is /-coupled to several neighbors.
/-coupling results from the magnetic interactions that occur when electrons contact nuclei, which
they do when they occupy molecular orbitals that have non-zero values at nuclear positions. For example,
electrons in a molecular orbitals contact both of the nuclei they help bond, but electrons in TT molecular
orbitals do not. Electrons are spin-V2 particles, and have intrinsic magnetic moments, just like spin-Vi
nuclei. If the spins of an electron and the nucleus it contacts have the same orientation, the orbital energy
of the electron will be slightly lower than it would be if their spins were antiparallel because of favorable
magnetic interactions. If electrons having both spin orientations contact a nucleus equally, the sum of
their interaction energies is zero.
Why does contact lead to splitting? Suppose two spin-V2 nuclei, A and B, are bonded by a molecular
orbital that contacts them both and contains two electrons, one spin up and the other spin down. If the
spin of A is parallel to the external magnetic field, electronic configurations that put the spin-up electron
close to A will be favored because they have lower energies. Since the electrons are paired, if the spin-up
electron is close to A, its spin-down partner must be close to B, and B will experience a small net
magnetic field because it is not "seeing" both electrons equally. If the orientation of the spin of nucleus
A were reversed, the bias in the spin orientation of the electron contacting nucleus B would also be
reversed, as would the magnetic field experienced by B. Thus within a population of identical molecules,
nuclei of type B will resonate at two slightly different frequencies, one for each of the two possible
orientations of the spin of A. The difference in resonant frequency between the two resonances is called
a splitting or a coupling constant, and splittings are mutual. The splitting of the resonance of B due to A
is the same as the splitting of the resonance of A due to B.
Four facts about /-coupling are relevant here. First, /-coupling effects are transmitted exclusively
through covalent bonds. Second, /-couplings between atoms separated by more than 3 or 4 bonds are
usually too small to detect. Third, splittings are independent of external magnetic field strength, and vary
in magnitude from a few Hz to 100 Hz in biological macromolecules, depending on the identities of the
atoms that are coupled, and the way they are bonded together. Fourth, macromolecular torsion angles
can be deduced from coupling constants because the magnitudes of three- and four-bond couplings
vary sinusoidally with torsion angle. Thus experiments that explore the couplings in a molecule's
spectrum can identify resonances arising from atoms that are near neighbors in its covalent structure, and
determine the magnitude of torsion angles.
1.2.4 Spin-Lattice Relaxation: Nuclear Overhauser Effects and Distances
Every resonance in an NMR spectrum has two times associated with it: a spin-lattice relaxation time,
or Ti, and a spin-spin relaxation time, or T2. Spin-lattice relaxation is important because a phenomenon
that contributes to it is an important source of information about interatomic distances. Spin-spin
relaxation is important in a negative way because it limits the sizes of the RNAs that can be studied by
NMR.
It takes time for the magnetic moments of nuclei to become oriented when a sample is placed in a
magnetic field, or to return to equilibrium, if their equilibrium orientations have been disturbed. Both pro
cesses proceed with first-order kinetics, and their rate constants are the same. The inverse of a first-order
rate constant is a time, of course, and in this case, that time is called the spin-lattice relaxation time, or T\.
Spin-lattice relaxation is caused by magnetic interactions that make pairs of neighboring nuclei in a
sample change their spin orientations in a correlated way. The rates at which such events occur depend
on a host of factors, among them the magnitudes of the magnetic moments of the atoms involved, the
external magnetic field strength, the distances between atoms, and the speed of their relative motions.
Everything else being equal, the slower a macromolecule rotates diffusionally, the longer the Ti-values of
its atoms. For RNAs the size of those being characterized by NMR today, proton spin-lattice relaxation
times range from 1 to 10 s.
Transmitters in modem spectrometers can be programmed to irradiate samples with pulses of elec
tromagnetic radiation that under favorable circumstances can instantaneously upset the spin orientation
of all the atoms in a molecular population that contribute to a single resonance, without disturbing
the orientations of any others. Suppose this is done to the Hl^ resonance of nucleotide n in some
A Spectroscopisfs View ofRNA Conformation: RNA Structural Motifs 5
RNA. What happens next? As the disequihbrated HI' population returns to equihbrium, exchanges of
magnetization that occur between its members and protons adjacent to them, cause the latter to "share"
in their disequilibrium. The H2' protons of nucleotide n are certain to be affected, as are nearby protons
belonging to nucleotide (n + 1). In molecules the size of an RNA, a reduction in the magnitude of
the resonances of adjacent protons results that becomes more pronounced with the passage of time out
to hundreds of milliseconds after the initial disequilibration, and then fades away. These changes in
resonance intensity are called nuclear Overhauser effects, or NOEs, for short.
NOEs are transmitted through space, and everything else being equal, their magnitude is inversely
proportional to the distance between interacting nuclei raised to the sixth power. In modern spectrom
eters, proton-proton NOEs, which are the ones usually studied, are large enough to measure if the
distance between nuclei is less than 5 A. Thus by studying NOEs, you can determine which protons are
within 5 A of any other proton in an RNA, and even estimate their separation.
1.2.5 Spin-Spin Relaxation: Molecular Weight Limitations
Spin-spin relaxation exists because NMR spectrometers detect signals only when the magnetic
moments of entire populations of nuclei are aligned, and moving in synchrony. This condition is met at
the outset of the typical NMR experiment, but as time goes on, the motions of the magnetic moments of
individual nuclei vary from the mean due to random, molecule-to-molecule differences in environments.
As the variation in the population grows, the vector sum of their magnetic moments decays to zero.
Since nuclear magnetic signals also lose intensity when individual nuclei return to their equilibrium
orientations, all processes that contribute to spin-lattice relaxation contribute to spin-spin relaxation
also. T2 is always shorter than T\.
NMR signals decay with first-order kinetics, and their characteristic times, Ti^, can be estimated by
measuring the widths of resonances in spectra. If T2 is short, resonances will be broad. If T2 is long,
resonances will be narrow. For RNAs in the molecular weight range of interest here, Tas are of the order
of 20 ms, and the more slowly a macromolecule tumbles, the shorter its T2. Thus big molecules have
broader resonances than small molecules.
The broadening of resonances that accompanies increased molecular weight contributes to the
difficulty of resolving the spectra of large RNAs. The chemical shift range over which RNA atoms
resonate is independent of molecular weight. Since large RNAs contain more atoms in chemically
distinct environments than small RNAs, the larger an RNA, the more resonances per unit chemical shift
there are in its spectra, on average, and the more difficult its spectra are to resolve. T2 broadening adds
insult to injury. The bigger the RNA, the broader its resonances, and broad resonances are harder to
resolve than narrow resonances. Since resolution of spectra is a sine qua non for spectroscopic analysis,
spectral crowding and resonance broadening combine to set an upper bound to the molecular weights of
the RNAs that can be studied effectively by NMR. The molecular weight frontier stands today (1999) at
about 45 nucleotides.
There is nothing permanent about this frontier. For example, the higher the field strength of a
spectrometer, the better resolved the spectra it produces. Thus as long as the field strengths of the
spectrometer magnets available continue to increase, as they have in the past, the frontier will continue to
move forward. The sensitivity improvement that accompanies increases in field strength is an important
added benefit of this very expensive approach to improving spectral resolution.
Isotopic labeling can also contribute. When multidimensional experiments are done on samples
labeled with ^^C and ^^N, spectra can be obtained in which proton resonances that have identical
chemical shifts are distinguished on the basis of differences in the chemical shifts of the ^^C or ^^N
atoms to which the protons in question are bonded, and hence /-coupled. Surprisingly, these techniques
have had a much bigger impact on NMR size limits for protein than they have for RNA. Proton T2'& in
macromolecules labeled with ^^C and ^^N are always shorter than those in unlabeled macromolecules
because of ^H-(^^C, ^^N) interactions, and the sensitivity of all experiments degrades as T2^ decrease.
For reasons that have yet to be fully articulated, this isotope-72 effect is more important in RNAs
than it is in proteins, and so in contrast to what protein spectroscopists have experienced, only modest
increases in the molecular weights of the RNAs that can be studied have resulted from the application
of heteronuclear strategies. What they have done is increase the reliability and completeness of the
assignments that are obtained for the spectra of RNAs of "ordinary" size.
6 A Spectroscopisfs View ofRNA Conformation: RNA Structural Motifs
RNA T2S can be reduced by selective deuteration because the relaxation rates of protons are
determined mainly by their interactions with neighboring protons. Thus when some protons in a
molecule are replaced with deuterons (^H), which have much lower magnetic moments, the relaxation
rates of the remaining protons decrease. Note that because deuterium resonates at frequencies well
outside the proton range, site-specific deuterium labeling can also be used to remove specific resonances
from the proton spectra of macromolecules, which can also help solve assignment problems (see below).
The molecular weight frontier is also being pushed forward by advances in experimental techniques
that do not depend on costly expedients like the construction of new instruments or complex isotopic
labeling schemes. The physics of relaxation in molecules containing several different kinds of magneti
cally active nuclei is a good deal more complicated than the description given above might lead one to
believe. By taking appropriate advantage of the opportunities this complexity affords, experiments can
be devised that produce macromolecular spectra similar to those less sophisticated experiments would
supply if T2S in samples were significantly longer than they really are (e.g. Pervushin et al^ and Marino
et alP). Novel experimental approaches like these, applied to isotopically labeled samples in ultra-high
field spectrometers, may make the analysis of 100-nucleotide RNAs possible in the next 5 years.
1.2.6 Samples
A single sample consisting of 0.2 ml of a 2 mM solution of an RNA can suffice for its structural
analysis. However, contrary to what is sometimes said, not all RNAs can be investigated under all
possible solvent conditions by NMR. A structure will not emerge from a spectroscopic investigation
unless the RNA of interest is monomeric under the conditions chosen, and has a single conformation. As
already suggested, it is sometimes convenient to study RNA samples that are labeled with ^^C, ^^N and
^H, either generally or site-specifically. Samples like this are not hard to make. The technology required
is constantly improving, and the cost continues to fall.^"*^
1.2.7 Multidimensional NMR
The modem era of macromolecular NMR began in the late 1970s, when the two-dimensional
spectra first began being obtained from proteins."* Among the first experiments done were the COSY
(or correlation Spectroscopy) and NOESY (or Nuclear Overhauser Spectroscopy) experiments. The
former generates a two-dimensional spectrum in which resonances that are 7-coupled are displayed, and
the latter does the same for resonances that cross-relax and hence give NOEs. The more complicated
multi-dimensional experiments introduced subsequently accomplish similar ends by different means.
Happily, there is not the slightest reason for the consumer of NMR structures to worry about the details.
1.2.8 Assignments
Ribonucleotides contain 8-10 protons of which 7-8 are bonded directly to carbon atoms, and hence
do not exchange rapidly with water protons. The remainder are bonded to nitrogens and oxygens, and
exchange rapidly. The resonances of an RNA's non-exchangeable and slowly exchanging protons can be
observed in spectra taken from samples dissolved in H2O, and the resonances of its non-exchangeable
protons can be studied selectively using samples dissolved in D2O. As Figure 1 shows, RNA resonances
cluster in four groups, depending on chemical type. Note, however, that the chemical shift separations
between groups of resonances are about the same size as environmental chemical shift effects that
disperse resonances within groups, and hence resonances can appear between clusters or even in the
"wrong" cluster. The ^^C and ^^N spectra of RNAs are similarly complex, but since the chemical
shift separation between groups is significantly larger (see Varani and Tinoco^^), "misplacement" of
resonances is less likely (but still not impossible).^^ An RNA's ^^P spectrum is always its worst dispersed
because all its phosphorus atoms appear in a single chemical context. Fortunately, there is only one
phosphorus resonance per residue.
The first order of business for the RNA spectroscopist is assignment of spectra, and this is invariably
the most time-consuming phase of any NMR project. A resonance is assigned when the atom (or
