J. Mol. Biol.

(1997) 269, 52±66

The Structure of an RNA ``Kissing'' Hairpin Complex

of the HIV TAR Hairpin Loop and its Complement
Kung-Yao Chang and Ignacio Tinoco Jr*

Department of Chemistry
University of California at
Berkeley and Structural Biology
Division, Lawrence Berkeley
National Laboratory, Berkeley
CA, 94720-1460, USA
*Corresponding author
We have used nuclear magnetic resonance (NMR) to obtain the structure
of an RNA ``kissing'' hairpin complex formed between the HIV-2 TAR
hairpin loop and a hairpin with a complementary loop sequence. Kissing
hairpins are important in natural antisense reactions; their complex is a
speci®c target for protein binding. The complex has all six nucleotides of
each loop paired to form a bent quasicontinuous helix of three coaxially
stacked helices: two stems plus a loop-loop interaction helix. Experimen-
tal constraints derived from heteronuclear and homonuclear NMR data
on 13C and 15N-labeled RNA led to a structure for the loop-loop helix
with an average root-mean-square deviation of 0.83 (0.10) A Ê for 33 con-
verged structures relative to the average structure. The loop-loop helix of
the kissing complex is distorted compared to A-form RNA. Its major
groove is blocked by the phosphodiester bonds that connect the ®rst loop
residue of each hairpin with its own stem, and it is ¯anked by two nega-
tively charged phosphate clusters. The loop-loop helix has alternating
helical twists between adjacent base-pairs. The base-pairs at the helix
junctions are overwound and three base-pairs near the helix junctions
adopt high propeller twists. All these changes reduce the distance needed
for the bridging phosphodiester bonds connecting each stem and loop to
cross the major groove of the loop-loop helix, and result in a deformed
RNA helix with localized perturbations in the minor groove surface. The
alternating helical twist pattern, plus other distortions in the loop-loop
helix may be important for Rom protein recognition of the kissing hair-
pin complex.
# 1997 Academic Press Limited
Keywords: NMR spectroscopy; RNA-RNA interactions; kissing hairpin
complex; alternating helical twist; RNA-protein recognition

Introduction plasmid control the initiation of DNA replication

Loop-loop interactions between RNA hairpins
participate in the antisense regulation of a variety
of cellular processes in prokaryotic cells and in bac-
teriophages (Wagner & Simons, 1994). Antisense
regulation is widespread in eukaryotes as well (for
example, see Pilipenko et al., 1996; Cavaille et al.,
1996). The most extensively studied biological sys-
tem that involves RNA loop-loop interactions is
the antisense regulation of ColE1 plasmid DNA
replication (Tomizawa et al., 1991 and references
therein). An RNA primer is used for the initiation
of DNA synthesis during replication of the ColE1
plasmid in Escherichia coli. The RNA primer is gen-
erated from an RNA precursor (RNAII) by RNase
H cleavage. A speci®c structure in RNAII is re-
quired for it to bind to the DNA near the replica-
tion origin, and to form the substrate for RNase H.
Two regulatory factors encoded by the ColE1
by interacting with RNAII to block the formation
of the speci®c structure required. One is an RNA
(RNAI) complementary to the 50 end of RNAII and
the other is a protein, Rom (RNA One Modulator),
that recognizes the RNAI-RNAII complex. The
complementary sequences of RNAI and the 50 end
of RNAII each can form three tandem stem-loop
structures with complementary loop sequences.
The interaction of RNAI and RNAII is initiated by
base-pairing between these complementary hairpin
loops through a kinetically favored intermediate,
the ``kissing hairpin complex''. The ®nal state is a
duplex formed by hybridization of the complemen-
tary RNAs. Rom speci®cally binds to the RNA
complex formed by two complementary hairpins
derived from RNAI and RNAII and stabilizes their
association (Tomizawa, 1990, 1993). It is the
speci®c loop-loop interaction, the kissing hairpin
complex, rather than the duplex that is recognized

0022±2836/97/210052±15 $25.00/0/mb971021 # 1997 Academic Press Limited

RNA Kissing Hairpins
by the Rom protein (Eguchi & Tomizawa, 1990,
1991).
Knowledge of the structures and stabilities of
RNA loop-loop interactions will be valuable for
understanding how RNAs interact with each other,
and how the folding of larger RNAs occur. NMR
and gel electrophoresis studies of the ColE1 kissing
hairpin complex (Marino et al., 1995) showed that
all seven nucleotides of the loop-loop helix formed,
and provided strong evidence that the complex is
bent. However, detailed structural information was
not obtained. Interactions between the kissing
hairpin complex and the Rom protein make this
system particularly attractive for studying RNA-
protein recognition (Predki et al., 1995) and pro-
tein-mediated RNA folding processes. One import-
ant question to ask is what are the structural
features of the kissing hairpin complex that are
recognized by the Rom protein. We had previously
studied the formation of a kissing complex be-
tween the TAR hairpin loop of HIV-2 (part of the
transcriptional activation region of the viral RNA)
and its complement (Chang & Tinoco, 1994). We
found that a 16-mer RNA hairpin (Tar*-16) with a
loop sequence (UCCCAG) complementary to the
TAR loop sequence (CUGGGA) associates speci®-
cally with the TAR hairpin loop (Tar-16) to form a
stable kissing hairpin complex. This complex can
be bound by the Rom protein with similar af®nity
to the wild-type kissing hairpin complex from the
ColE1 plasmid (Chang, 1995). Here, we describe
the solution structure of the Tar-Tar* RNA kissing
hairpin complex. The loop-loop interaction helix
contains unique structural features that may be a
general characteristic of kissing hairpin complexes
recognized by the Rom protein. Loop-loop inter-
actions are important in folding of RNAs and
make likely sites for metal ion and protein binding.
Results
Spectral assignment and characterization of
secondary structure features
Our previous study (Chang & Tinoco, 1994) had
shown that the Tar-Tar* kissing hairpin complex
involved the formation of at least ®ve base-pairs
between the two hairpin loops as shown in
Figure 1. The imino proton resonances of all 11
possible G
C base-pairs and three of ®ve possible
A
U base-pairs were seen in the exchangeable pro-
ton NMR spectrum of the Tar-Tar* complex. The
formation of A*12
U*5 for the Tar*-16 in the complex,
and the loop-loop A11
U*6 base-pair could not be
established because of the absence of U*5 and U*6
imino protons in the 1D spectra, presumably due
to fast exchange with solvent.
To obtain a high-resolution structure of the Tar-
Tar* kissing complex we used unlabeled samples,
plus samples in which one of the hairpins in the
complex was 13C-labeled and the other unlabeled.
The absence of H10 -H20 sugar proton crosspeaks in
a DQF-COSY spectrum (Marion & WuÈthrich, 1983)
53
Figure 1. A representation of the secondary structure of
Tar-Tar* based on exchangeable and non-exchangeable
proton NMR data. The intra-strand and cross-strand
AH2-H1 NOE network of the ®ve adenine residues of
Tar-Tar* are indicated by arrows.
of the complex indicated that all the sugar puckers
of the residues were in a 30 -endo conformation, as
expected for an A-form RNA duplex. The ®ve ade-
nine H2 protons were distinguished from the H6/
H8 protons by their very different 13C chemical
shifts in a natural abundance 13C HMQC exper-
iment (Varani & Tinoco, 1991b). Three adenine H2
protons were assigned to the A2 of Tar-16 and A*10,
A*14 of Tar*-16 from the strong NOE seen between
the imino proton of the A
U base-pair and the H2
of the adenine residue.
A 2D 2H2O NOESY spectrum of the complex
was very crowded with few crosspeaks easily
identi®ed. However, the spectrum was greatly sim-
pli®ed by isotope-editing of an RNA sample with
one hairpin 13C-labeled and the other hairpin left
unlabeled. We used nucleotides speci®cally 13C-la-
beled to simplify the spectrum and to reduce the
broadening effects introduced by uniformly labeled
nucleotides (SantaLucia et al., 1995). Figure 2(a)
and (b) are the H10 -H6/H8 portion of a 13C-1/2 ®l-
tered NOESY spectrum (Otting & WuÈthrich, 1990)
of the complex with all 16 residues of Tar-16
speci®cally 13C-labeled at the C6 (pyrimidines) or
C8 position (purines) of the bases. Figure 2(a) is
the sub-spectrum containing the H10 -H6/H8 NOE
that involves 13C-attached protons and corresponds
to the Tar-16 part of the complex. There are two
H10 -H6/H8 NOE connectivity walks in Figure 2(a)
that correspond to the NOE connectivity of two
fragments of stacked RNA oligonucleotides. One is
a 5-mer with a 50 to 30 base sequence of Pur-Pur-
Pur-Pyr-Pyr and the other is an 11-mer with a base
sequence of Pyr-Pyr-Pur-Pur-Pur-Pur-Pur-Pur-Pyr-
Pyr-Pyr. The six sequential purines in the 11-mer
segment indicate that this segment is C6UGG-
54
Figure 2. H10 -H6/H8 region of 2H2O NOESY spectra of
Tar-Tar*. (a) Two H10 -H6/H8 NOE connectivity walks
in the 13C sub-spectrum from a 300 ms 13C 1/2-®ltered
NOESY spectrum of Tar-Tar* with C6/C8 13C-labeled
Tar-16. They correspond to nucleotides in Tar-16 and
lead to a secondary structure that has all six loop resi-
dues stacking on the 30 side of Tar-16 stem. (b) Two
H10 -H6/H8 NOE connectivity walks in the 12C sub-spec-
trum from a 300 ms 13C 1/2-®ltered NOESY spectrum
of Tar-Tar* with C6/C8 13C-labeled Tar-16. They corre-
spond to nucleotides in Tar*-16 and lead to a secondary
structure that has all six loop residues stacking on the 30
side of Tar*-16 stem.
RNA Kissing Hairpins
GAGGCUC16 and thus helps assign the 5-mer seg-
ment to G1AGCC5. Similarly, in the NOESY sub-
spectrum that originated from 12C-attached H6/H8
protons (Figure 2(b)), two NOE connectivity walks
belonging to a 5-mer and an 11-mer are assigned as
the G*1C*U*G*U*5 and U*6C*C*C*A*G*A*C*A*G*C*16
of the Tar*-16 in the complex.
3D HMQC-NOESY experiments (Fesik &
Zuiderweg, 1990) on the complex with either hair-
pin fully labeled with 13C-enriched C6/C8 NTPs
were used in the assignment of non-anomeric
sugar protons. A short mixing time 2H2O NOESY
experiment on unlabeled complex was used to as-
sign the H20 sugar protons through intra-ribose
H10 -H20 and internucleotide H20 -H6/H8 NOEs.
This information, plus the previously assigned H10
and base protons, help to assign each H20 sugar
proton of the complex. An H20 -aromatic proton
NOE connectivity walk for the loop residues of the
Tar*-16 part of the complex is presented as an as-
sembly of 2D slices taken from the 3D HMQC-
NOESY spectrum (Figure 3). In A-form RNA, the
H20 , H30 sugar protons are less than 4 A Ê away
0
from the H5 base proton of an adjacent 3 pyrimi-
dine (Varani & Tinoco, 1991a). This property was
used to con®rm the H20 proton assignments as
well as the assignment of 15 H30 protons. H10 -H30
proton NOEs were then used to con®rm these as-
signments and to locate the remaining H30 protons.
An H30 -H6/H8 NOE connectivity walk for the
same region of loop residues of Tar*-16 is shown
in Figure 3 and is consistent with the previous 10
and 20 -H6/H8 NOE connectivity walks. The sugar-
aromatic proton NOE connectivity patterns and
the observations of aromatic-aromatic proton
NOEs on adjacent residues (data not shown)
suggest continued stacking within these four frag-
ments of RNA (two 5-mers and two 11-mers). The
identi®cation of two non-interrupted 11-mers indi-
cates that the continuing stacking of loop residues
extends into the 30 side of both hairpin stems.
Two extra crosspeaks (boxed) in Figure 2(a) and
(b) suggest that intra-hairpin NOEs exist between
the C5H10 and C6H6 of Tar-16, as well as between
the U*5H10 and U*6H6 of Tar*-16. However, because
of spectral overlap, alternative interhairpin connec-
tivities are possible. Similar ambiguity occurs in
tracing the H20 /H30 -aromatic proton NOE connec-
tivities at the same stem ±loop junction. Therefore,
13
C double-1/2-®ltered NOESY experiments
(Otting & WuÈthrich, 1990) on a sample containing
uniformly labeled Tar-16 and unlabeled Tar*-16
were used to distinguish the intrahairpin from in-
terhairpin NOEs. The non-anomeric sugar proton-
aromatic proton region of a 13C-edited NOESY
spectrum showed that there is no intra-hairpin
U*5H20 -U*6H6 NOE in Tar*-16 in a 12C double-se-
lected NOESY spectrum compared with the same
region of a NOESY spectrum of an unlabeled com-
plex. In contrast, an inter-hairpin NOE between the
C5H20 of Tar-16 and the U*6H6 of Tar*-16 as well as
an inter-hairpin NOE between the U*5H10 , H20 of
Tar*-16 and the C6H6 of Tar-16 can be detected in
RNA Kissing Hairpins
Figure 3. H20 and H30 -H6/H8 connectivity walk on 2D
slices of 3D HMQC-NOESY of Tar-Tar* with Tar*-16
13
C-labeled at C6/C8 position. It shows the 20 -walk
(black line) and 30 -walk (gray line) from U*6 to G*15 of
Tar*-16 and further supports a secondary structure with
30 -stacking of all the loop residues. The number shown
in the right corner within each slice is the 13C chemical
shift of each corresponding C6 or C8 carbon atom.
a 12C-selected, 13C-®ltered double-1/2-®ltered
NOESY spectrum (data not shown). These inter-
hairpin NOEs combined with the previous H10 /
H20 /H30 -H6/H8 NOE connectivity walks ®x the
orientation of the loop-loop helix. They support the
secondary structure shown in Figure 1, with con-
tinuous stacking of the 30 -side of each stem into
each loop. However, the ®rst base of each loop
(nucleotide 6 of each hairpin) stacks on the term-
inal residue of the 50 side of the stem of the other
hairpin (nucleotide 5 of each hairpin). It does not
stack on its own stem.
In an A-form helix, each adenine H2 proton has
an intra-strand NOE interaction with the H10 pro-
ton of the ribose on its 30 side as well as a cross-
strand AH2 to H10 NOE to the ribose of the
55
Figure 4. AH2-H10 NOEs of the ®ve adenine residues of
the complex in the 12C sub-spectrum from a 300 s 13C
1/2-®ltered NOESY spectrum of Tar-Tar* with C6/C8
13
C-labeled Tar-16. Two unusually strong AH2-H10
NOEs (labeled in bold face) are seen. These crosspeaks
have similar intensities to pyrimidine H5-H6 crosspeaks;
they are the only adenine crosspeaks seen at 60 ms mix-
ing times.
base-pairing partner of its 50 nearest neighbor
(Varani & Tinoco, 1991a). The AH2-H10 proton
NOE spectra show that every adenine residue in
the complex has two AH2 to H10 proton NOEs
(Figure 4). This indicates that all the adenine resi-
dues are surrounded by a duplex environment, in-
cluding A*12 of Tar*-16 and A11 of Tar-16 (Figure 1),
which also have interhairpin AH2-H10 NOEs with
the H10 of C6 in Tar-16 and C*7 in Tar*-16, respect-
ively. Thus, although the imino protons of the po-
tential A11
U*6 and A*12
U*5 are not seen in the
exchangeable proton spectrum, their characteristic
AH2-H10 NOE patterns indicate that these adenine
residues are in a double helix environment. These
data also indicate the existence of a continuous
helix starting from the end of the Tar-16 hairpin
stem and ending at the end of the Tar*-16 hairpin
stem with the loop-loop interaction helix in be-
tween.
There are two unusually strong AH2 to H10
intra-strand NOEs in the short mixing time
NOESY spectrum. They occur between A11H2 and
G12H10 of Tar-16, and between A*10 H2 and G*11 H10
of Tar*-16. These unusual intrastrand AH2-H10
NOE intensities are very different from those
expected from a normal A-form RNA helix and
suggest an unusual conformation in the minor
groove of the loop-loop helix. Examination of the
imino-H10 crosspeak intensities of a 100 ms mixing
time H2O NOESY also reveals the existence of rela-
tively strong G8NH-G9H10 and G10NH-A11H10
NOEs, which are much weaker in a normal A-form
helix (data not shown).
56
Phosphorus chemical shifts can be used to locate
unusual phosphate backbone torsion angles in nu-
cleic acids (Gorenstein, 1981). Several phosphorus
chemical shifts outside the normal phosphorus
chemical shift region of an A-form RNA were seen
in a 31P-1H heteronuclear COSY spectrum (Sklenar
et al., 1986) with a span of 2.2 ppm for the phos-
phorus chemical shift range (data not shown). Un-
fortunately, attempts to make a sequence-speci®c
assignment of these phosphorus resonances
through a 2D hetero-TOCSY-NOESY experiment
(Kellogg et al., 1992) were not successful, probably
because of the large size of the complex.
The unassigned non-anomeric sugar protons-
H6/H8 NOEs in the 3D HMQC-NOESY spectra
were picked as potential candidates for the intra-
nucleotide H40 and H50 , H500 -H6/H8 NOEs. They
are assigned to H40 , H50 or H500 if corresponding
H10 -H40 or H10 -H50 /H500 proton NOEs can also be
found. Because the 13C chemical shift of C4 and C5
of sugars are very different, they help to dis-
tinguish and con®rm the H40 and H50 /H500 proton
assignments available through 13C HMQC and
constant-time HSQC spectra (Vuister & Bax, 1992).
This led to the assignment of most of the H40 , H50 /
H500 sugar protons of the Tar*-16 and some H40 of
Tar-16.
Structure determination
Experimental constraints and
structure calculations
Figure 5 shows a diagram summarizing the in-
ternucleotide NOE network observed in the
NOESY spectra of the complex. A total of 423 NOE
distance constraints, 60 experimental dihedral con-
RNA Kissing Hairpins
straints, and 39 hydrogen bond constraints were
used for the structure calculations (Table 1); this
corresponds to 16 experimental constraints per
residue. Weak A-form constraints were used for
the ®rst two base-pairs of Tar* and the ®rst three
base-pairs of Tar. One hundred random structures
were generated using X-PLOR (BruÈnger, 1990) and
were then subjected to a simulated annealing-re-
strained molecular dynamics protocol followed by
an energy minimization step. The minimization
steps were carried out without including electro-
static interactions. The total energy and the NOE
violation energy of each structure were used as cri-
teria for identi®cation of converged structures.
Figure 6 shows the calculated structures arranged
in order of increasing total and NOE violation
energies. There is a small jump in the total energy
after the ®rst 39 structures, and a jump in NOE
violation energies after 33 structures. The six struc-
tures in between did not form the A*
U* base-pair
at the end of the Tar*-16 stem (Figure 1); these
structures had short interproton distances not seen
as NOE crosspeaks. We therefore chose the ®rst 33
structures for further analysis. A second set of
structural constraints that included hydrogen bond
constraints for A11
U*6 and A*12
U*5 was used for
similar structure calculations. These constraints led
to a higher convergence ef®ciency with 44 struc-
tures with low total energy and NOE-violation en-
ergy generated from 100 random structures.
However, the average structure and the precision
of the range of structures for both sets of con-
straints were very similar. We are convinced that
all base-pairs in the complex form, although the
two U imino groups from the junction region are
in fast exchange with solvent.
Figure 5. The inter-nucleotide NOE
network of the Tar-Tar* kissing
hairpin complex. The two hairpins
are arranged in duplex form with
the shaded rectangular bars repre-
senting the presence of the imino
protons in the 1D exchangeable
proton spectrum. Inter-hairpin
NOE bonding constraints were
added only for base-pairs that had
imino protons visible in the spectra.
Inter-hairpin NOEs occur at the
helix junctions (C5-U*6 and U*5-C6),
and in the loop-loop helix; 16
NOEs for exchangeable protons are
not shown.
RNA Kissing Hairpins
Table 1. Statistics of experimental constraints used in the structure calculations, and precision
of the lowest-energy structures
A. Distance and dihedral constraints
Intraresidue
Interresidue
Intrahairpin
Interhairpin
Hydrogen bond
Dihedral constraints
Constraints per residue
B. Precision of lowest-energy structuresa
Whole complex (16 bp)
One-turn (11 bp; C4-G13, U*3-A*14)
Loop-loop interaction helix (6 bp)
C6-A11, U*6-G*11
a
The root-mean-square deviation (RMSD) of the 33 lowest-energy structures from the average structure.
The precision of the ®nal calculated structure
was measured by comparing the root-mean-square
deviation (RMSD) of the 33 converged structures
from the average structure (Table 1). The RMSD
for the stem regions of Tar-16 and Tar*-16 are 0.55
(0.13) and 0.58 (0.11) A Ê , respectively, and the
loop-loop interaction helix has a calculated RMSD
of 0.83 (0.10) AÊ . Using all atoms in the complex,
an RMSD of 1.71 (0.33) A Ê relative to the average
structure was obtained. This variation is mainly
caused by different global features among the con-
verged structures. There are 26 bent structures of
different bending angle (from 30 to 90 ) and seven
linear structures among the 33 converged struc-
tures. No obvious energy differences or NOE viola-
tions distinguish these two different global
structures. It is well known that accurate global
conformations of elongated nucleic acids can be
very dif®cult to obtain from NMR data. Thus, we
concentrate on the local structural features that can
be determined well by NMR. Shown in Figure 7(a)
Figure 6. Structures plotted as a function of increasing
NOE-violation energies (®lled circles) and increasing
total energies (®lled squares).
57
197 Ê)
Strong (1.8± 3.0 A 70
226 Medium (2.0 ±4.0 A Ê) 150
198 Weak (3.0 ±5.0 AÊ) 164
28 Very weak (4.0 ±7.0 A Ê) 39
39
60
16
RMSD (A Ê)
1.71  0.33
1.28  0.19
0.83  0.10
is the superposition of the central part of the 22
lowest energy structures (RMSD of 1.28 (0.19) A Ê
calculated for 33 converged structures). They
correspond to two and three base-pairs of the
stems of Tar-16 and Tar*-16, respectively, as well
as the loop-loop interaction helix. The remaining
terminal base-pairs in each stem are not included
because their proton chemical shift values remain
unchanged upon complex formation. The RMSD
value suggests that the local geometry is relatively
well de®ned within each helical region. The heli-
cal parameters of the 33 converged structures
generated from the different sets of constraints
were analyzed using the program CURVE
(Lavery & Sklenar, 1989). The three parameters
(twist, roll and tilt) that de®ne the relative orien-
tation of successive base-pairs (Dickerson et al.,
1989) are listed in Table 2. They are precisely
determined with standard deviations that are
within 10% of the mean value for most of the
values. A representative structure is shown in
Figure 7(b).
Structural features of the Tar-Tar* kissing
hairpin complex
The formation of a new helix of six base-pairs
involving all the residues within the loops and the
requirement to maintain the covalent linkage in the
stem ±loop junction of each hairpin imposes severe
constraints on the 16 base-pair Tar-Tar* kissing
hairpin complex. Shown in Figure 8 is a side-by-
side comparison of the central part (11 bp) of the
complex with one turn of standard A-form RNA
and B-form DNA (Saenger, 1984) of identical se-
quences (with thymine in the DNA). The path of
the phosphate backbone in the junction region
where the three helices of the complex stack is a
very distinct structural feature of this kissing hair-
pin complex. The continuous 30 -stacking of the
loop residues that extend into the 30 -side of each
hairpin stem create a gap between U*5 and U*6 as
well as between C5 and C6 that can be connected
58 RNA Kissing Hairpins

Figure 7. (a) Superposition of the central 11 base-pairs of the Tar-Tar* complex (six from the loop-loop helix, three
from the Tar* stem and two from the Tar stem) for 22 converged structures with lowest total energies. The protons
are omitted to allow a clearer presentation of the backbones. (b) Stereo view of a single low-energy structure.

most easily by crossing the major groove of the
loop-loop interaction helix. These gaps are about
Ê long for A-form geometry and need at least
10 A
two nucleotides to cross them (Haasnoot et al.,
1986). In the three-dimensional structure of the
Tar-Tar* complex, only one phosphodiester bond is
used to connect each gap (Figure 9(a)). The phos-
phodiester bonds that cross the major groove to
bridge the ®fth and sixth residues in each hairpin
are found to be pushed toward each other, thus
blocking accessibility of the major groove formed
by the loop-loop helix (Figure 9(b)).
Short interphosphorus distances are observed
between the 50 phosphate group of C5 and the 50
phosphate groups of U7 and G8 of Tar-16
(5.25(0.77) AÊ and 9.25(1.21) A Ê , respectively) as
well as between U*5, and C*7, C*8 of Tar*-16
(6.7(0.9) AÊ and 7.2(1.3) A
Ê , respectively). In con-
trast, the corresponding interphosphorus distances
in an A-form RNA helix are 10.4 A Ê and 12.7 AÊ.
This results in two sets of spatially close, nega-
tively charged phosphate clusters located at the
edges of the major groove (Figure 9(a)). One set is
the 50 phosphate groups of C5, C6, U7 and G8 of
RNA Kissing Hairpins 59

Table 2. Helical parameters between successive base-pairs in the loop-loop interaction helix
and in both hairpin stems from 33 lowest-energy structures
Base-pair step Twist Roll Tilt
Loop-loop helix:
U*6-A11 to C*7-G10 35.0  5.3 21.2  9.0 5.1  8.0
C*7-G10 to C*8-G9 27.8  3.8 13.1  2.1 3.3  6.0
C*8-G9 to C*9-G8 42.2  4.2 29.3  6.6 10.6  12.6
C*9-G8 to A*10-U7 33.2  3.4 ÿ5.2  4.3 ÿ3.0  5.0
A*10-U7 to G*11-C6 40.9  3.5 7.0  4.6 ÿ14.0  5.4
Average: 35.8  2.8 0.3  1.8

Tar-16 stem:
G1-C16 to A2-U15 33.6  3.0 6.1  8.4 8.0  7.1
A2-U15 to G3-C14 26.4  2.1 4.6  5.2 ÿ1.3  9.4
G3-C14 to C4-G13 29.1  2.3 26.1  9.5 ÿ6.8  7.7
C4-G13 to C5-G12 32.1  3.2 22.4  11.5 ÿ0.7  5.0

Tar*-16 stem:
G*1-C*16 to C*2-G*15 33.1  1.6 13.5  3.8 11.5  4.7
C*2-G*15 to U*3-A*14 29.2  1.4 6.8  3.2 ÿ9.7  3.1
U*3-A*14 to G*4-C*13 30.5  2.1 ÿ3.7  5.9 2.3  3.3
G*4-C*13 to U*5-A*12 22.1  2.1 6.6  5.2 2.3  3.3

Tar-16 and the other set is the 50 phosphate
groups of U*5, U*6, C*7 and C*8 of Tar*-16. The in-
terphosphorus distances in the Tar* phosphate
cluster are very similar to those between the 50
phosphate groups of U8, A9, C11 and U12 of yeast
tRNAPhe that form a Mg2‡-binding site (Holbrook
et al., 1977). These two phosphate clusters ¯ank-
ing the major groove of the loop-loop interaction
helix may constitute part of a speci®c metal ion-
binding site. The Tar-Tar* complex (Chang &
Tinoco, 1994) and the wild-type ColE1 kissing
hairpin complex (Tomizawa, 1990; Gregorian &

Figure 8. Comparison of the central 11 base-pairs of the Tar-Tar* complex with standard B-form DNA and A-form
RNA. The red residues correspond to the stem of Tar*-16 and the violet residues belong to the stem of Tar-16. The
pink and blue residues in the middle are the loop residues of Tar*-16 and Tar-16, respectively.
60
Figure 9. (a) View into the major groove of the Tar-
Tar* kissing hairpin complex with the ¯anking phos-
phate clusters shown as yellow spheres. (b) Comparison
of the loop-loop helix with the same sequence of stan-
dard A-RNA looking down from the top of the major
groove. It shows the blocking of the major groove of the
loop-loop helix by the two crossing phosphodiester
bonds. The arrows indicate the phosphate groups of C6
of Tar-16 and U6* of Tar*-16.
Crothers, 1995) are speci®cally stabilized by mag-
nesium ions.
There is base stacking continuing from the A-
form stems through the entire RNA complex with
some distortions within the loop-loop helix and
overwinding at the interhairpin stacking steps be-
tween the bases of C5 and U*6, and between U*5 and
C6. Speci®cally, three base-pairs located at the helix
junctions are found to adopt very high propeller
twist. They are C6
G*11, A11
U*6 and U*5
A*12, and
their propeller twist values are 38(4) , 30(14)
and 26(7) , respectively, with one of them shown
in Figure 10. These high propeller twists can im-
RNA Kissing Hairpins
Figure 10. The high propeller twist of the A*12
U*5
base-pair in the helical junction.
prove the stacking between the purines in the
junction regions and may make important contri-
butions to the stability of the complex.
Table 2 shows that the twist angle between adja-
cent base-pairs along the loop-loop helix varies al-
ternately from the average twist angle of the helix.
An overtwist in one step is followed by an under-
twist in the next step. The two central base-pairs of
the loop-loop helix have an average twist angle of
42(4) for the step from G8
C*9 to G9
C*8. Away
from this central step, there is an undertwist step
and an A-form step (average 28(4) and 33(3) )
that are followed by another two overtwist steps
(average 35(5) and 41(4) ). This leads to an
average twist angle of 36(3) in each step
(Table 2).
The unique structural features ®t well with the
NMR data, especially the unusual AH2/NH-H10
NOE intensities in the short-mixing time 2H2O/
H2O NOESY spectra (Figure 4). These intensities
within the loop-loop interaction helix are very im-
portant characteristics for this distorted helix and
can be used to distinguish it from the normal A-
form RNA helix. These NOEs occur in the base-
pairs adopting the high propeller twist and alter-
nating helical twist and are an indication of a
narrow minor groove. There are several unusual
aromatic proton chemical shifts in residues near
the two terminal base-pairs of the loop-loop helix.
They are the unusually down®eld-shifted H6 of C6
in Tar-16 (8.51 ppm) and H6 of U*6 (8.65 ppm), and
unusually up®eld-shifted H10 (5.01 ppm), H8
(6.65 ppm) of G*11 in Tar*-16. These unusual chemi-
cal shifts can be qualitatively explained by the
stacking patterns found in the structure. The
down®eld H6 chemical shift of the two pyrimi-
dines in the sixth position of both hairpins can be
rationalized by the deshielding of both H6 protons
caused by the destacking. In contrast, the position
RNA Kissing Hairpins
Figure 11. Minor groove water-accessible surface of the
loop-loop helix of the Tar-Tar* complex compared with
the minor groove surface of a standard A-form RNA
duplex of the same sequence. The surfaces are generated
using a water probe of 1.4 AÊ radius on Insight II (Bio-
sym Technologies Inc.) with the colors red, blue, yellow
and green representing O1P, O2P, phosphorus and the
hydrogen atoms of 20 -hydroxyl groups, respectively.
of the purine ring of A*10 can explain the up®eld
G*11 H8 chemical shift.
The average structure of the kissing complex is
bent towards the major groove (although with a
large variation in bending angles among the
converged structures) as seen in Figure 7(b). The
main contributions to the bend are the large posi-
tive roll angles in the loop-loop helix, particularly
at the center step (Table 2). Together, the over-
wound loop-loop helix and the bend in the center
of the helix combine to reduce the distance for the
phosphodiester bonds to cross the major groove of
the helix. The loop-loop interaction helix also pro-
duces a narrower minor groove with localized sur-
face perturbations compared with a normal A-form
helix. This perturbed minor groove surface can be
characterized by the base-pair tilts and twists
(Table 2), and by cross-strand C10 -C10 distances.
For example, the C10 -C10 distances from U7 to
61
G*11 and A*12 are 7.9(0.4) A Ê and 7.2(0.3) AÊ , re-
spectively. The corresponding distances in an A-
form helix are 8.7 AÊ and 9.3 A
Ê . Figure 11 is a view
of the minor groove surface of the loop-loop inter-
action helix compared with a normal A-form RNA
helix of the same sequence. There is a clear differ-
ence in the water-accessible surfaces, and the phos-
phodiester backbone in the loop-loop interaction
helix is pushed toward the minor groove. The spa-
cings between phosphate groups and the 20 -hy-
droxyl groups create distinct features within the
minor groove surface. This perturbation may be a
general feature of the kissing hairpin complex, and
is consistent with the distinct RNase V1 cleavage
pattern in the ColE1 kissing complex compared
with a duplex of the same sequence (Eguchi &
Tomizawa, 1990).
Discussion
The helix formed by base-pairing between the
two hairpin loops in the kissing complex is signi®-
cantly different from an A-form helix. The main
NMR data that determine its structure are: (1) H10 ,
H20 , H30 -aromatic NOE connectivities that require
continuous stacking of bases throughout the com-
plex. (2) Cross-strand AH2-H10 NOEs indicating
formation of the A
U pairs at the ends of the loop-
loop helix. (3) An unusual pattern of NOE intensi-
ties that are consistent with an alternate overwind-
ing and underwinding in the loop-loop helix,
overwinding at the junctions between the helices,
and a high propeller twist of the base-pairs at the
junctions (Figure 10). All these structural features
are well characterized by the precision of the range
of structures consistent with the NMR data. Large
positive roll angles and large (but varied) tilts are
found for the base-pairs along the loop-loop helix
(Table 2). The large standard deviations observed
in the tilt angle of the central step is indeed caused
by the existence of two conformers with very
different tilt angles (0 versus 25 ). The average
structure of the kissing complex is bent. However,
there was a wide range of bend angles; 26 struc-
tures had bending angles ranging from 30 to 90 ,
and seven were approximately linear. Bending can
also be interpreted as anisotropic ¯exibility (Crick
& Klug, 1975) in this center region of the loop-loop
interaction helix.
Gel electrophoresis and NMR data showed that
the ColE1 kissing hairpin complex is bent towards
the major groove with an estimated bending angle
of 45 (Marino et al., 1995). All seven bases in the
loop-loop helix were paired and continuous stack-
ing of the loop bases on the 30 -side of each stem
occurred. It is encouraging that the two kissing
complexes, although containing different sequences
and loop lengths, have qualitatively similar struc-
tures.
Sequence-dependent, local structure variations
are well documented in DNA, such as in alter-
nating B-DNA (Klug et al., 1979; Yuan et al.,
62
1992) and A-tract DNA (Nelson et al., 1987; Yoon
et al., 1988), and may be important in a variety
of transcription regulation processes involving
DNA-protein interactions (Calladine & Drew,
1992). For example, sequence-dependent alternat-
ing helical twist is involved in the met repressor-
operator complex (Somers & Phillips, 1992) and
in the complex between telomeric DNA and the
DNA-binding domain of RAP1 (KoÈnig et al.,
1996). Also, DNA twisting on the non-contacted
bases of the bacteriophage 434 operator has been
shown to affect the af®nity of the bacteriophage
434 operator for bacteriophage 434 repressor
(Koudelka et al., 1987, 1988; Koudelka & Carlson,
1992).
In contrast to DNA, RNA helices are found to be
more rigid (Egli et al., 1996). We thus think that the
structural variations seen in the loop-loop helix are
caused by the constraints imposed by the presence
of the three coaxially stacked helices. For example,
analysis of the phenylalanine tRNA structure did
not reveal sequence-speci®c structure variation
(Holbrook et al., 1981). Our analysis of available
RNA crystal structures also suggests that the alter-
nating helical twist pattern observed here is not a
result of a sequence-dependent effect. There are
two CUG steps in the complex: one in the stem of
Tar* and one in the loop-loop helix. The two helical
twist angles in the C*U*G* step of the Tar* stem
are found to have similar value of 30 compared
with the overtwist, undertwist pattern seen in the
loop-loop interaction helix of the identical CUG
step (Table 2). This pattern remains unchanged
when structures are calculated with the presence of
electrostatic interaction in the ®nal energy mini-
mization stage. The helix distortions seen in the
Tar-Tar* kissing complex suggest that deformabil-
ity may be important for the formation of a stable
complex. The sequence requirement for the for-
mation of a kissing hairpin complex may thus be
the exclusion of sequence combinations that lead to
reduced deformability (Gregorian & Crothers,
1995).
Distortion of RNA helices has been found to be
a major contribution for RNA recognition. It can be
achieved by either the insertion of a bulge into an
RNA helix as found in the HIV TAR hairpin
(Puglisi et al., 1992; Aboul-ela et al., 1995), or the
formation of a mismatch within a helix, as seen in
the HIV RRE internal loop (Battiste et al., 1996). In
both cases, the distortion of an RNA helix leads to
the opening of the major groove and thus allows
sequence-speci®c RNA recognition. Both the P1
substrate recognition by the core of Tetrahymena
group I intron (Doudna et al., 1989; Strobel & Cech,
1995) and the acylation of the tRNAAla acceptor
stem by tRNAAla synthetase (Gabriel et al., 1996)
require a distorted RNA helix with a conserved
G
U wobble base-pair for recognition. The recog-
nition of the kissing hairpin complex by the Rom
protein represents another type of protein recog-
nition of an RNA helix. A fully Watson-Crick-
paired helix is recognized, but it is distorted by its
RNA Kissing Hairpins
context between two adjacent helices. The distor-
tion is presumably induced by the constraints im-
posed by the maintenence of 30 -helical stacking of
the loop residues, plus the A-form geometry of the
hairpin stems.
Rom binding
For the hybridization of RNAI and RNAII to
proceed, the base-pairs from the helices of the hair-
pins need to melt; this is a very slow process
(Turner et al., 1990) without the help of helicases. It
was shown that the Rom protein can affect the kin-
etics of hybridization between RNAI and RNAII
by decreasing the dissociation rate of the kissing
hairpin complex, but there is no evidence that the
Rom protein is acting as a helicase. Because the
Rom protein can bind to kissing hairpin complexes
of very different sequences, it suggests that com-
mon structural features shared among different
complexes are recognized by Rom. The unique fea-
tures of the central loop-loop helix of the Tar-Tar*
kissing complex include: a compressed major
groove, two groups of four closely spaced phos-
phate groups around the major groove, and a dis-
torted minor groove surface. The loop-loop helix
distortion is characterized by successive steps of
over- and underwinding, and increased propeller
twists for the junction base-pairs. Presumably, all
these structural characteristics contribute to Rom
recognition. Further studies on other kissing hair-
pin complexes of different loop sequences and loop
size will be needed to address this problem.
Materials and Methods
Preparation of RNA sample
C6/C8 13C-labeled NMPs were chemically synthesized
(SantaLucia et al., 1995) and were gifts from Dr John San-
taLucia Jr. Uniformly 13C-labeled NMPs were isolated
from Methylophilus methylotrophus bacterial cells supplied
by the Stable Isotope Resource at Los Alamos National
Laboratory following the published procedures (Batey
et al., 1992; Nikonowicz et al., 1992). The 13C-labeled
NMPs were converted into their NTP forms by enzy-
matic phosphorylation (Haynie & Whitesides, 1990).
NMR quantities of RNA samples were synthesized
in vitro using phage T7 RNA polymerase with a T7 pro-
moter and speci®c DNA templates. The transcription
yield of isotope-labeled RNA was optimized on an ana-
lytical scale by varying the concentration of Mg2‡ and
then scaled up to a 20 ml transcription reaction for the
preparation of RNA for NMR studies. The concen-
trations of Tar-16 and Tar*-16 RNA were measured at
280 nm because of their high G ‡ C contents.
NMR spectroscopy
RNA samples were dialyzed against 50 mM NaCl at
pH 6.4 (H2O sample) or 180 mM NaCl at pH 7.0 (2H2O
sample) in 0.01 mM EDTA, 10 mM sodium phosphate
buffer. The ®nal sample concentrations were 1 to 1.5 mM
for the 1D NMR studies and 2 to 2.5 mM for 2D or 3D
homonuclear and heteronuclear NMR studies. The vo-
RNA Kissing Hairpins
lumes of NMR samples were between 0.4 and 0.5 ml for
the unlabeled sample and 0.2 to 0.25 ml for the labeled
samples. All the heteronuclear NMR experiments were
done on a Bruker AMX 600 spectrometer except for the
2D 31P-1H heteronuclear correlation experiment, which
was measured on a Bruker AMX 300 spectrometer. All
NMR data were acquired using the time-proportional
phase incrementation (TPPI, Marion & WuÈthrich, 1983)
or States-TPPI (Marion et al., 1989) formats and then
transferred to a Silicon Graphics workstation and pro-
cessed by the Felix 2.30 program (Biosym Technologies
Inc.).
The experimental constraints used in the determi-
nation of structure were all taken from NMR data with-
out magnesium present. However, 1D and 2D
exchangeable and non-exchangeable proton NMR data
in the presence of 5 mM Mg2‡ or 180 mM Na‡ showed
no signi®cant changes in spectra, such as unusual chemi-
cal shifts or changes in AH2 to imino or H10 NOE inten-
sities.
The H2O NOESY spectrum of 2.5 to 3 mM RNA in
50 mM NaCl at pH 6.4 at 3 C were measured with 100
to 300 ms mixing times using a 1-1 NOESY pulse se-
quence (Sklenar & Bax, 1987) with the carrier frequency
set on the water resonance. In all, 2K complex points in
t2 and 300 to 350 t1 increments were acquired with 96
scans per t1 increment and spectral width of 12,000 Hz
on a Bruker 600 spectrometer. Prior to the Fourier trans-
formation, the data sets were processed by digital shift
subtraction in both dimensions and apodized using a 30
skewed sine bell in the ®rst dimension and 60 in the
second dimension; the data were zero ®lled in both di-
mensions to give a ®nal 2K  2K real data matrix.
The 2D double-quantum-®ltered COSY spectra (DQF-
COSY, Marion & WuÈthrich, 1983) and 2H2O NOESY
spectra (Macura et al., 1982) at 60 to 300 ms mixing time
were recorded at 25 C with a low power preirradiation
during a two second relaxation delay to suppress the re-
sidual H2O peak. The 2K complex points in t2 and 350 to
400 (FIDs) in t1 were collected with a spectral width of
4608 Hz (500 MHz spectrometer) or 5000 Hz (600 MHz).
A proton-detected heteronuclear 31P-1H COSY was re-
corded as proposed by Sklenar et al. (1986) with a spec-
tral width of 600 Hz in the 31P dimension and 1400 Hz in
the 1H dimension. 118 FIDs of 2K complex points were
collected with 176 scans per FID on a Bruker AMX 300
spectrometer. A natural abundance proton-detected
13
C-1H HMQC spectrum (Bax et al., 1983) was recorded
with a spectral width of 7550 Hz in the 13C dimension
having the 13C carrier set at the resonance frequency be-
tween that of C5 and C6 of the bases. This leads to the
out-of-phase folding of the adenine C2 resonances and
all non-anomeric carbon resonances in ribose into an
empty space of the spectrum (Schmieder et al., 1991). A
GARP1 composite sequence (Shaka et al., 1985) for broad
band carbon decoupling was applied during acquisition
and a refocusing delay of 2.5 ms was set for optimal exci-
tation of aromatic resonances. Exponential multiplication
was applied with 3 and 5 Hz linewidth broadening in t2
and t1, respectively, during data processing. 13C-1H
HMQC and HSQC (Bax et al., 1990; Vuister & Bax, 1992)
spectra were also taken on the complex with C6/C8 or
uniform 13C-labeling of either hairpin with an acquisition
time of 2.5 hours.
13
C-editing NOESY experiments were done on C6/C8
or uniformly 13C-labeled samples with a spectral width
of 5000 Hz in both dimensions and 1K complex
points using the pulse sequences suggested by Otting &
WuÈthrich (1990). The delay 1/4J was tuned to 1.25 ms
63
for ef®cient excitation of aromatic resonances
(JCH ˆ 200 Hz). The 3D HMQC-NOESY spectra (Fesik &
Zuiderweg, 1990) were taken on the complex with C6/
C8 13C-labeled Tar-16 or Tar*-16 with a delay 1/2J set to
3.3 ms for ef®cient excitation of the sugar resonances
(JCH ˆ 150 Hz). The carbon carrier was set to the middle
of the carbon sugar spectrum with a spectral width of
6250 Hz (o1), 1500 Hz (o2) and 1200 Hz (o3). The total
number of increments in every dimension was t1 ˆ 32,
t2 ˆ 64 resulting in 2048 FIDs and the ®nal real matrix
after zero-®lling contained o1 ˆ 512  o2 ˆ 128  o3 ˆ
64 points.
NOE distance constraints
The 2D 2H2O NOESY and 13C-®ltered NOESY exper-
iments of 60, 90, 120 and 200 ms mixing time were
done on unlabeled and labeled samples. The NOE vo-
lumes of all well-isolated crosspeaks from spectra of
different mixing times were estimated and compared
with crosspeaks of constant distances in order to obtain
distance constraints for non-exchangeable protons. To
alleviate the ambiguity of crosspeak measurement
caused by crosspeak overlap for residues in some cru-
cial junction regions, 13C-labeled C6/C8 NTPs were
speci®cally incorporated into the complex. For example,
13
C-labeled C6/C8 UTPs were incorporated into Tar*-
16 to help distinguish U5, U6 and C7 in the stem± loop
junction. NOE crosspeaks were classi®ed as strong,
medium, weak and very weak. NOE crosspeaks that
can be seen at 60 ms mixing times with crosspeak in-
tensities similar to those of pyrimidine H5-H6 cross-
peaks were classi®ed as strong and were assigned an
inter-proton distance range of 1.8 to 3.0 A Ê . Nearly all
(90%) of the strong crosspeaks are intranucleotide H10 -
H20 and internucleotide H20 -H6/H8 crosspeaks. Those
NOEs not found at 60 ms mixing times but that ap-
peared at 90 ms mixing times were grouped as med-
ium and given a distance range of 2.0 to 4.0 A Ê. A
distance range of 3.0 to 5.0 A Ê was assigned for NOE
crosspeaks classi®ed as weak that were absent in short
mixing time NOESYs but occurred at 120 ms mixing
times. Finally, NOE crosspeaks that are seen only at
200 ms or longer mixing times were put into the group
of very weak with an assigned inter-proton distance of
4.0 to 7.0 AÊ . Fixed distances as suggested by Saenger
(1984) were also assigned between atoms involved in
hydrogen bond formation in Watson-Crick base-pairs
as indicated by exchangeable proton NMR data. The
100 ms H2O NOESY data were used to estimate the
inter-proton distances that involve the exchangeable
protons, using imino-AH2 crosspeaks as reference.
Imino-imino proton distances between base-pairs were
set based on the result from 300 ms H2O NOESY con-
nectivity walks. In total, 423 experimental distance con-
straints involving non-exchangeable and exchangeable
protons were obtained from observed proton-proton
NOEs.
Dihedral constraints
A 30 -endo conformation was assigned to all the ribose
units of the complex based on the absence of H10 -H20
crosspeaks in a DQF-COSY spectrum and was achieved
by restraining the endocyclic torsion angles u0, u1, u2
and u3. The glycosidic torsion angles w of A11, U*5, U*6
and A*12 were left randomized due to the absence of U
imino exchangeable proton NMR data. The glycosidic
torsion angle w was set to be anti for all other
64
nucleotides, because of the lack of strong H10 -H6/H8
NOE.
For the other ®ve backbone dihedral angles, a set of
loose A-form helix constraints (Saenger, 1984) were used
to constrain the backbone of residues in the ®rst two
(Tar*) or three (Tar) base-pairs of the stem of each
hairpin with a range of 20 for torsion angles b, g, e
and a range of 30 for torsion angles a, z. No non-ex-
perimental dihedral constraint was used to restrain the
other residues in each hairpin: C4 to G13 in Tar16 and U*3
to A*14 in Tar*16. A set of arti®cial constraints was also
used to keep the amino groups of the bases in the same
plane as the aromatic ring. A total of 60 experimental di-
hedral constraints and 72 loose A-form helical constraints
were used.
Molecular dynamics
Structure calculation on the complex was done by re-
strained molecular dynamics followed by energy mini-
mization using the X-PLOR program (BruÈnger, 1990)
with a force-®eld consisting of bond lengths, bond
angles, improper angles, repulsive van der Waals poten-
tial and the experimental distance and dihedral angle
constraints from the NMR data. Random structures were
created with random backbone torsion angles and used
as the initial structures for a two-stage structure calcu-
lation. First, a modi®ed simulated annealing protocol
(Wimberly, 1992) was used to search for global fold
structures that are consistent with secondary structure
features determined by NMR using only NOE distance
constraints. The global fold structure was then subjected
to the second stage structure re®nement that included all
the dihedral angle constraints in addition to the NOE
distance constraints. Dihedral angle constraints were
added gradually: ®rst b, g, d, e and then a and z. This
structure re®nement procedure did not change the global
shapes of calculated structures. The converged structures
were selected based on low NOE-violation energies and
total energies of the structures (Figure 6). These con-
verged structures were then subjected to the ®nal stage
of energy minimization with attractive Lennard-Jones
potentials introduced to obtain proper van der Waals
contacts.
The program CURVES (Lavery & Sklenar, 1989) was
used to analyze the helical parameters of the structure.
Acknowledgements
We thank the Stable Isotope Resource at Los Alamos
National Laboratory for providing cells used in isolation
of the uniformly 13C/15N-labeled NMPs, and Dr John
SantaLucia Jr for the synthesis of C6/C8 labeled 13C
NMP. We thank Professor Lynne Regan and Dr Paul
Predki at Yale University for their kind gift of the plas-
mid encoding the Rom protein. It is a pleasure to thank
Mr David Koh for synthesizing all the DNA oligonucleo-
tides used throughout this work, Dr Jeffrey Pelton for
help with the NMR experiments and Ms Barbara Den-
gler for managing the laboratory. We appreciate the criti-
cal reading of this manuscript and useful suggestions
from Dr Gabriele Varani. This research was supported in
part by National Institute of Health grant GM 10840, by
the Department of Energy grant DE-FG03-86ER60406,
and through instrumentation grants from the Depart-
RNA Kissing Hairpins
ment of Energy (DE-FG05-86ER75281) and from the
National Science Foundation (DMB 86-09305).
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