doi:10.1006/jmbi.2000.3730 available online at http://www.idealibrary.com on
J. Mol. Biol. (2000) 299, 103±112
Crystal Structure of an Adenine Bulge in the RNA
Chain of a DNA RNA Hybrid,
d(CTCCTCTTC) r(gaagagagag)
Chellappanpillai Sudarsanakumar, Yong Xiong
and Muttaiya Sundaralingam*
Biological Macromolecular
Structure Center, Departments
of Chemistry, Biochemistry and
Biophysics Program, The Ohio
State University, 012
Rightmire Hall, 1060 Carmack
Road, Columbus
OH 43210, USA
Crystal structure of a DNA RNA hybrid, d(CTCCTCTTC) r(gaagagagag),
with an adenine bulge in the polypurine RNA strand was determined at
Ê resolution. The structure was solved by the molecular replacement
2.3 A
method and re®ned to a ®nal R-factor of 19.9 % (Rfree 22.2 %). The hybrid
duplex crystallized in the space group I222 with unit cell dimensÊ , b ˆ 47.61 A
Ê and c ˆ 54.05 A
Ê , and adopts the A-form
ions, a ˆ 46.66 A
conformation. All RNA and DNA sugars are in the C30 -endo conformation, the glycosyl angles in anti conformation and the majority of the
C40 -C50 torsion angles in g‡ except two trans angles, in conformity with
the C30 -endo rigid nucleotide hypothesis. The adenine bulge is looped out
and it is also in the anti C30 -endo conformation. The bulge is involved in
a base-triple (C g)*a interaction with the end base-pair (C9 g10) in the
minor groove of a symmetry-related molecule. The 20 hydroxyl group of
g15 is hydrogen bonded to O2P and O50 of g17, skipping the bulged adenine a16 and stabilizing the sugar-phosphate backbone of the hybrid. The
hydrogen bonding and the backbone conformation at the bulged adenine
site is very similar to that found in the crystal structure of a protein-RNA
complex.
# 2000 Academic Press
*Corresponding author
Keywords: X-ray structure; A-form conformation; hybrid; adenine bulge;
looped out
Introduction
Single and multiple bulges are one of the frequently occurring secondary structural elements
providing structural ¯exibility required for RNA
folding (Wyatt et al., 1993). Bases that lack a pairing partner in the opposite strand of the double
helix are known as bulges, which are potentially
signi®cant in RNA tertiary folding (Woese &
Gutell, 1989) and provide sites for speci®c proteinRNA interactions (Moine et al., 1997; Valegard et al.,
1997). An extra base could stack into the double
helix or loop out and can cause misalignment in
the helix axis. Bulges may introduce a signi®cant
destabilization of DNA and RNA duplexes (Patel
et al., 1982; Morden et al., 1983; Woodson &
Present address: C. Sudarsanakumar, School of Pure
and Applied Physics, Mahatma Gandhi University,
Kottayam, Kerala 686 560, India.
E-mail address of the corresponding author:
[email protected]
0022-2836/00/010103±10 $35.00/0
Crothers, 1989; LeBlanc & Morden, 1991). Solution
NMR studies on DNA and RNA show a stacked-in
conformation of single adenine bulges (Hare et al.,
1986; Nikonowicz et al.,1989; 1990; Rosen et al.,
1992; Borer et al., 1995), while single pyrimidine
bulges show stacked-in or looped-out conformations or a temperature-dependent equilibrium
between the two (Morden et al., 1990; van den
Hoogen et al., 1988a; Kalnik et al., 1989, 1990).
Bulged adenine is a common feature in many ribosomal RNAs (Gutell et al., 1985), self-splicing
group I, group II and nuclear pre-mRNA introns
(Cech, 1990; Moore et al., 1993; Wittop Koning &
Schumperli, 1994). Looped-out adenine bulges are
speci®c sites for RNA-protein recognition, as found
in the binding sites of Escherichia coli ribosomal
RNA-protein complexes, 5 S rRNA to L18 and 16 S
rRNA to S8 (Peattie et al., 1981; Moine et al., 1997)
and in the binding of bacteriophages R17 and
MS2 coat proteins to the translational operator
fragments of their replicase gene (Valegard et al.,
1997).
# 2000 Academic Press
104
Figure 1. The numbering scheme for the hybrid
duplex. DNA nucleotides are given in uppercase letters
and RNA in lowercase.
Relatively very little is known about bulges,
especially bulges in hybrid duplexes. Structural
studies on DNA/RNA hybrids with bulges are signi®cant due to their applications in anti-sense technology, by a possible cleavage of the RNA strand
within the duplex at bulge sites in the presence of
metal ions (Husken et al., 1996; Hall et al., 1996).
Small nucleolytic ribozymes also undergo selfcleavage in the presence of metal ions. A convenient folding of the RNA chain is essential
to provide a suitable in-line geometry for their
catalysis (Lilley, 1999). The crystal structure of a
homopyrimidine homopurine DNA RNA hybrid,
d(CTCCTCTTC) r(gaagagagag) with an adenine
bulge (underlined) was analyzed to understand the
conformation of the bulge and also the effect of the
bulge on the conformation of the hybrid duplex.
The polypurine RNA strand of this hybrid is an
analogue of the polypurine tract in human immunode®ciency virus 1 (HIV-1) genome, which is
unaffected by RNase-H (Cof®n, 1996).
Results and Discussion
Conformation of the hybrid duplex
The numbering scheme for the nonamer hybrid
bulge is shown in Figure 1, deoxyribonucleotides
in one strand are designated by uppercase letters
and ribonucleotides in the other, in lowercase. The
hybrid duplex adopts the standard A-form conformation (Figure 2). All the deoxyribose and ribose
sugar rings are in C30 -endo conformation, the C40 C50 exocyclic torsion angles are g‡ (except for a12
and g17) and the glycosidic torsion angles are anti.
The average values for the pseudo rotation phase
angles of the sugar are 21 and 15 , for the DNA
and RNA strands, respectively. Helical parameters
were calculated using the program CURVES and
are given in Table 1. The minor groove width
Ê to 11.7 A
Ê . The major groove
ranges from 10.9 A
width, measured by the only P-P distance due to
the limited length of the regular nonamer duplex,
Ê.
is 7.2 A
Conformation of the bulge
The bulged adenine is looped out of the helix.
The sugar moiety of the bulge is ¯ipped over such
that it is almost perpendicular to the helix axis
(Figure 2(b)). Single adenine bulges are in loopedout conformation in crystal structures of DNA
Adenine Bulge in RNA of a DNA RNA Hybrid
(Miller et al., 1988; Joshua-Tor et al., 1992), DNARNA chimer (Portmann et al., 1996), RNA (Ennifar
et al., 1999) and RNA-protein complexes (Valegard
et al., 1997). Looped-out conformation for single
adenine bulges in A-form DNA and RNA were
also shown to be favorable by energy minimization
studies (Zacharias & Sklenar, 1999). Also, the conformation of the bulge is strongly in¯uenced by
the backbone conformation of the ¯anking nucleotides. The bulged base in this structure is in anti
conformation (ÿ166 ), the sugar moiety is in the
C30 -endo pucker and the C40 -C50 exocyclic torsion
angle is in the g‡ conformation.
Effect of bulge on backbone conformation
It is known that bulges can introduce destabilization in DNA and RNA duplexes, and the extent
of destabilization depends on many factors such as
the length of the bulge loop (Longfellow et al.,
1990), the nature of the bulge base and the ¯anking
residues (LeBlanc et al., 1991). The ¯anking bases
on the 50 and 30 sides of the bulge in this structure
Ê and a twist of 29 .
are stacked with a rise of 3.5 A
The accommodation of a looped-out residue in the
RNA strand produces a change in the sugar-phosphate backbone conformational angles around
the bulge. The phosphodiester torsion angles on
the 50 and 30 sides of the bulge, a(g17) ˆ 47 and
z(g15) ˆ ÿ 156 and the adjacent torsion angles (e
on the 50 side and b and g on the 30 side) show distortion (Table 2). The RNA sugar-phosphate backbone conformation at the bulged nucleotide is
stabilized by hydrogen bonds between the 20
hydroxyl group of g15 and O50 and O2P atoms of
g17, by skipping the bulge a16 (Figure 3). The P-P
Ê;
distances involving the bulge (P15-P16: 5.29 A
Ê
P16-P17:5.03 A) are shorter than the average P-P
Ê ) in the RNA strand. This shortening
distance (5.7 A
of the P-P distances causes unusually close spacing
between the phosphate oxygen atoms (O2P(g15)Ê ) on the 50 side of the bulged
O2P(a16) ˆ 4.21 A
base. A similar conformation and hydrogen bonding pattern were observed for the sugar-phosphate
backbone at the bulged adenine site (a10) in the
crystal structures of two protein-RNA complexes,
between recombinant MS2 virus capsids and their
RNA operator fragments (Valegard et al., 1997)
(Figure 4) and also, for the looped-out single adenine bulge (a210) in the P4-P6 domain of the group
I intron (Cate et al., 1996). In the Mg-form of the
11-mer (Portmann et al., 1996), short distances
between adjacent phosphate oxygen atoms were
observed on the 30 side of the bulged adenine and
a spermine molecule is coordinated to these oxygen atoms. In the crystal structure of group I
intron, short distances between phosphate oxygen
atoms were also observed in the adenine-rich
bulge in the P5a helix, where the phosphate oxygen atoms are coordinated to two magnesium ions
(Cate et al., 1996). Also, in the crystal structure of
the HIV-I TAR RNA fragment, the ucu bulge-phosphate oxygen atoms show short distances where a
105
Adenine Bulge in RNA of a DNA RNA Hybrid
Figure 2. Structure of the
d(CTCCTCTTC) r(gaagagagag) hybrid showing the bulge. (a) Skeleton of the molecule superimposed
on the 2Fo ÿ Fc map at 1s level.
(b) Superposition of ®nal atomic
coordinates of the hybrid bulge
(thick line) with the coordinates
of
an
analogue
hybrid,
d(CTCTTCTTC) r(gaagaagag).
bound Ca2‡ stabilizes the bulge conformation
(Ippolito & Steitz, 1998). In these examples the
negatively charged regions involving the closely
spaced phosphate oxygen atoms are potential sites
for the binding of metal ions. In the present structure no cations are bound, but a cation binding site
is possible on the 50 side of the bulged base
between the phosphate oxygen atoms of g15 and
a16. One water molecule is observed in this site at
hydrogen bonding distances from the phosphate
oxygen atoms of a16.
In the present structure, all the residues in the
DNA and RNA strands, including the bulged residue are in the C30 -endo conformation. The looped-
out bulge did not alter the sugar pucker of the
neighboring residues both in the RNA strand and
the opposite DNA strand. In the spermine form of
the 11-mer; the bulge sugar, the two preceding
sugars and one sugar opposite to the bulge in the
other strand show deviations from the standard
C30 -endo sugar pucker while, in the Mg-form the
sugar preceding the bulge alone shows deviation
(Portman et al., 1996).
Effect of bulge on groove dimensions
A superposition of the structure of this hybrid
duplex without the bulge and a similar analogue
hybrid
duplex,
d(CTCTTCTTC)r(gaagaagag)
Table 1. Helical parameters of d(CTCCTCTTC) r(gaagagagag) calculated with the program CURVES
Residue
Twist
Rise
Inclination
dx
Propellor twist
1C-g
2T-a
3C-g
4C-g
5T-a
6C-g
7T-a
8T-a
9C-g
Average
26.0
34.5
29.0
28.1
34.5
32.0
27.6
34.4
30.8
2.9
3.1
3.5
2.9
2.7
2.8
3.3
3.2
3.1
1.6
2.4
2.6
4.1
5.7
6.3
6.2
6.0
7.5
4.7
ÿ4.4
ÿ4.4
ÿ4.1
ÿ4.3
ÿ4.2
ÿ4.5
ÿ3.9
ÿ4.3
ÿ4.1
ÿ4.3
7.1
ÿ4.0
ÿ9.3
ÿ7.7
ÿ11.5
ÿ8.2
ÿ17.0
ÿ15.9
ÿ13.9
ÿ8.9
106
Adenine Bulge in RNA of a DNA RNA Hybrid
Table 2. Backbone and glycosyl torsion angles of d(CTCCTCTTC) r(gaagagagag)
Residue
a
b
g
d
e
z
w
P
A. DNA
C1
T2
C3
C4
T5
C6
T7
T8
C9
Average (SD)
ÿ61
ÿ56
ÿ63
ÿ58
ÿ69
ÿ63
ÿ59
ÿ84
ÿ64(8)
160
164
170
170
174
163
169
185
169(7)
ÿ15
59
54
56
50
51
61
64
55
56(5)
88
81
82
84
81
81
82
95
91
85(5)
ÿ145
ÿ150
ÿ156
ÿ149
ÿ151
ÿ148
ÿ151
ÿ176
ÿ153(9)
ÿ79
ÿ75
ÿ80
ÿ78
ÿ76
ÿ76
ÿ81
ÿ58
ÿ75(7)
ÿ175
ÿ163
ÿ160
ÿ163
ÿ156
ÿ1 60
ÿ161
ÿ155
ÿ146
ÿ160(7)
6
18
17
13
21
23
20
36
38
21(10)
B. RNA
g10
a11
a12
g13
a14
g15
a16
g17
a18
g19
Average (SD)
ÿ77
154
ÿ54
ÿ63
ÿ50
92
47
ÿ67
ÿ58
ÿ65(8)
185
210
157
168
166
ÿ164
105
172
161
169(10)
46
58
175
47
58
54
56
173
56
54
53(5)
84
83
82
73
74
84
86
79
78
75
78(4)
ÿ165
ÿ163
ÿ124
ÿ133
ÿ150
ÿ111
ÿ124
ÿ139
ÿ143
ÿ151(12)
ÿ64
ÿ81
ÿ96
ÿ68
ÿ84
ÿ156
100
ÿ80
ÿ83
ÿ76(8)
ÿ166
ÿ162
ÿ179
ÿ170
ÿ166
ÿ154
ÿ166
178
ÿ168
ÿ165
ÿ166(2)
11
15
11
12
18
16
7
19
16
26
16(5)
The values which deviate more than 1 SD are considered outliers and are underlined. a12 (italics) and g15, a16 and g17 (bold) are
not included in the calculation of Average and SD. The bulge residue (a16) and the ¯anking residues (g15 & g17) show deviation in
their backbone conformational angles. The end base-pair (C9g10), which is involved in the base-triple interaction and the residues
(C1, T8, a-11, a12 and g13) in the abutting interaction region show deviation in their backbone conformational angles. a12 is in the
trans,trans (a,g) conformation, the only one of this kind observed in this structure.
SD, Standard deviation.
determined in this laboratory (Xiong &
Sundaralingam, 2000) show that they compare
Ê and the
favorably with an overall rmsd of 0.90 A
deviations are predominant in the RNA strand at
the bulge site (Figure 2(b)). Therefore, the double
helical part of this structure is affected very little
by the bulge. In comparison with the above hybrid,
the measured kink angle is 6 , which is much
smaller than that reported for single base bulges
(Lilley, 1995). The deviations of the phosphate
groups on either side of the bulged base, P16 and
P17, result in a widening of the major groove and
the ¯anking base atoms lie exposed and are accessible to interacting molecules like proteins. The
crystal structure of the 11-mer (Portmann et al.,
1996) and NMR studies in solution (Puglisi et al.,
1995, Ye et al., 1995) also supports the widening of
the major groove.
Figure 3. Stereo plot showing the intramolecular hydrogen bonds between O20 of g15 and O2P and O50 of g17 stabilizing the RNA back bone conformation of the molecule at the bulge site. Hydrogen bonds are marked as broken
lines. The possible direction of attack by the bulge 20 oxygen atom at the adjacent phosphate group on the 30 side of
the bulge is indicated by the continuous line. The O20 -P distance and O20 -P-O50 angle are marked.
Adenine Bulge in RNA of a DNA RNA Hybrid
107
Figure 4. Stereo plot showing the comparison of the hybrid bulge structure (RNA in green and DNA in pink) with
the wild-type RNA fragment (light blue) of the protein-RNA complex. Two base-pairs on both sides of the bulged
residue are shown.
Base-triple interaction involving the
bulged adenine
The bulged adenine forms a base-triple,
(C9 g10)*a16 in the minor groove with the end
base-pair (C9 g10) of a symmetry-related molecule
(Figure 5). The three bases are almost in the same
plane. The base atom, N1 of a16, accepts an interÊ ) from the 20 -OH
molecular hydrogen bond (2.6 A
group of g10. Four symmetry-related molecules
form two base-triples (Figure 5(a)) and they stack
on each other in such a way that the bulged out
Figure 5. Stereo plot showing the base-triple interaction. (a) Interaction among four symmetry-related molecules
forming two base-triples between the bulged adenine and the end base-pair (C9 g10) are shown. Bulged adenine (yellow), DNA chain (pink) and RNA chain (green). (b) Projection of two symmetry-related base-triples that stack on
each other, perpendicular to their base planes. a16 shown (yellow), C9 (pink) and (light pink) and g10 (green) and
(light green). * Indicates residues from symmetry-related molecules. Watson-Crick hydrogen bonds (red broken lines).
The hydrogen bond between N1 of a16 and O20 of g17 is shown as a white dotted line.
108
Adenine Bulge in RNA of a DNA RNA Hybrid
Figure 6. Stereo plot showing the abutting interactions. (a) Abutting interaction of the end base-pair (C1 g19) in
the minor groove of a symmetry-related molecule. DNA strand (pink) and RNA strand (green). (b) A close view of
abutting interactions (white broken lines). DNA residues C6-C9 (pink) and C1-T2 (light pink) and RNA residues g10g13 (green) and a18-g19 (light green) are shown. Intermolecular hydrogen bonds (yellow broken lines). * Indicates
residues from symmetry-related molecules.
adenine stacks on the base-pair (C9 g10) with the
sugar ring and the base of a16 over the base of C9
and g10, respectively (Figure 5(b)). Further, a
water molecule provides additional stability to the
stacking by bridging a16 and C9 through hydrogen
bonds. The bulged-out adenine bases are also
involved in base-triple interactions, (c g)*A in the
minor groove of both crystal forms of the 11-mer
(Portmann et al., 1996).
Abutting interactions
One of the terminal base-pairs (C1 g19) is
involved in abutting interactions in the minor
groove of a symmetry-related molecule (Figure 6),
while the other (C9 g10) is not; it is involved in a
base-triple interaction as discussed above. The base
atoms of C1 and g19 stack over the sugar atoms of
T7 and T8, respectively. The RNA residues a12 and
g13 and the DNA residues T7, T8 and C9 are
involved in intermolecular hydrogen bonds with
the terminal base-pair (C1 g19) as shown in
Figure 6(b). The O20 of g19 makes two intermolecular hydrogen bonds with O20 and N3 of (a12)
Ê for both) providing additional stability in
(ˆ2.6 A
this region. The abutting interactions and the presence of the above strong hydrogen bonds may be
the reason for the distorted extended trans, trans
(a,g) backbone conformation adopted by a12. The
abutting interaction is the unique feature observed
in this structure. Thus, hydrogen bonding, van der
Waals and stacking interactions provide a tight
packing environment for the hybrid bulged
duplex.
Interactions of O20
All the 20 -hydroxyl groups except that at a16 and
g17 are involved in intramolecular and/or intermolecular hydrogen bonds. The 20 -hydroxyl
groups at the 50 and 30 ends of the RNA strand are
involved in intermolecular hydrogen bonds stabilizing the base-triple and the abutting interactions,
respectively. The O20 of g15 form intramolecular
hydrogen bonds with O50 and O2P of g17 providing a stable conformation to the sugar-phosphate
backbone at the bulge site. There are two intramolecular hydrogen bonds between a 20 hydroxyl
group of the RNA residue and the sugar atom O40
of the next 30 residue (between 20 OH groups of g10
and a18 with O40 of a-11 and g19, respectively).
The hydroxyl group at a14 is connected to its base
atom, N3, through water-mediated hydrogen
bonds. There are two O20 -O50 intramolecular
hydrogen bonds, connecting O20 at g10 and a-11
with O50 at a-11 & a12, respectively.
109
Adenine Bulge in RNA of a DNA RNA Hybrid
Table 3. Conformation of single adenine bulge in crystal structures
Nature of
the duplex
Various forms (no of
independent bulges)
Conformation of the
bulge
A-form
Hybrid
A-form
Chimer
A-form
RNA
(1)
Looped-out
This structure
Mg-form (1)
Sp-form (1)
Trigonal (2)
Portmann et al. (1996)
(CGCGAAATTTACGCG)2
B-DNA
(2)
(CGCAGAATTCGCG)2
B-DNA
(2)
A-form
RNA
Pro-flavin
Soaked form (2)
Wild-type (1)
C5-complex (1)
Looped-out
Looped-out
Looped-out
Looped-out
Looped-out
Looped-out
Looped-out
Looped-out
Looped-out
Stacked-in
Looped-out
Stacked-in
Looped-out
Looped-out
Sequence
(CTC-CTCTTC)
(gagagagaag)
(gcgATATAcgc)2
(cuugcugaggugcacacagcaag)2
acaugagga
uguac-cc
Monoclinic (2)
Reference
Ennifar et al. (1999)
Miller et al. (1988)
Joshua-Tor et al. (1992)
Valegard et al. (1997)
Bulged residue in bold and underlined.
The paired stem of the RNA bulge-loop in the RNA-protein complex.
a
Hydration
There are 23 crystallographically independent
water molecules in the structure. Of these, ten are
found in the major groove and one in the minor
groove, all of them are involved in hydrogen
bonds with the hetero atoms of the bases. Among
these ten major groove water molecules, two of
them are hydrogen bonded to the DNA bases and
the remaining to the RNA bases. There are nine
water molecules involved in hydrogen bonds with
the phosphate oxygen atoms.
Biological significance
Knowledge of the conformational preferences of
bulges is essential to understand RNA folding. The
adenine bulge in this hybrid duplex (¯anked by
guanine bases, gag), is in the looped-out conformation. All the crystal structures on single adenine
bulges given in Table 3 show looped-out conformation. The only exception to this is the stacked-in
adenine in one of the strands in the 13-mer DNA.
This also shows a stacked-in conformation in solution (Nickonowicz, et al., 1989, 1990). Therefore
the looped-out conformation for a single adenine
bulge is most prevalent in the solid state, irrespective of the crystal packing. The RNA-protein
(Valegard et al., 1997) complexes in crystals also
show a looped-out conformation for single adenine
bulges (gag) even though the RNA itself in solution studies reveal a stacked-in conformation (Wu
& Uhlenbeck, 1987; Borer et al., 1995). E. coli ribosomal protein-rRNA complexes in solution also show
a looped-out conformation for single adenine
bulges (Peattie, et al., 1981; Moine et al., 1997). Solution studies and energy minimization calculations
on small DNA and RNA oligonucleotides containing gag show stacked-in conformation (van den
Hoogen et al., 1988b; Zacharias et al., 1999). The
conformational similarity of this hybrid structure
with the crystal structures of the RNA-protein
complexes mentioned above (both contain gag)
reveals that the crystal packing interactions involving the bulged adenine (as found in this structure)
or the interactions with molecules like proteins (as
found in RNA-protein complexes) could be a
deciding factor for the looped-out conformation in
the solid state. The lack of such well-de®ned interactions may be a reason for the stacked-in conformation observed in solution studies and in energy
calculations.
In single bulge structures, the RNA self-cleavage
occurs via an attack of the 20 -OH group of the
bulge nucleotide at the phosphate group on the 30
side, resulting in a 20 ,30 -cyclic phosphate group at
the bulged residue and a free 50 -hydroxyl terminus
on the next residue (Brown et al., 1985; Lilley, 1999;
Portmann et al., 1996; Husken et al., 1996). The calÊ)
culated values for O20 (a16)-P(g17) distance (4.3 A
and O20 (a16)-P(g17)-O50 (g17) angle (124 ) show
that the 20 -O atom of the bulged sugar is oriented
towards the scissile P(g17)-O50 (g17) bond on the 30
side of the bulged adenine for a possible hydrophilic attack (Figure 3). A similar conformation is
observed in the modeled Mg-form crystal structure
of the 11-mer at the 30 side of the bulged adenine
Ê
with O20 -P distance and O20 -P-O50 angle 3.8 A
and 145 , respectively (Portmann et al., 1996).
A remarkable difference is that the negatively
charged potential formed by the short distances
between the phosphate oxygen atoms is observed
on the 30 side of the bulge in the 11-mer, while in
the present structure it is located on the 50 side of
the bulged residue. Also, the O2P atom of the scissile phosphate and the O50 atom of the scissile
bond are hydrogen bonded to the 20 -OH group of
the ¯anking residue on the 50 side of the bulge in
this structure. It is known that in the presence of
divalent metal ions, small nucleolytic ribozymes
catalyze a site-speci®c transesteri®cation reaction
in which the 20 hydroxyl group attacks the 30 phos-
110
Adenine Bulge in RNA of a DNA RNA Hybrid
phate group. The most important factor for the
occurrence of their cleavage is the correct folding
of the RNA in order to satisfy the active geometry
(Lilley, 1999). It is also known that the cleavage
activity is possible for hammerhead, hairpin and
Neurospora VS ribozymes in the presence of extremely high concentrations of monovalent metal ions
(Murray et al., 1998). In the present structure, the
conformation of the RNA strand at the bulge site
appears to be favorable for catalysis but there is no
clear evidence for the involvement of a metal ion.
Methods
Synthesis and crystallization
The deoxyribonucleotide d(CTCCTCTTC) of the allpyrimidine DNA and the decaribonucleotide r(gaagagagag) of the all-purine RNA were synthesized by solid
phase phosphoramidite chemistry using an in-house
automated nucleic acid synthesizer. The oligomers were
puri®ed by ion-exchange chromatography and ethanolprecipitation. Separate strands of DNA and RNA were
mixed in 1:1 ratio at a single-strand (DNA/RNA)
concentration of 2 mM, incubated at 363 K for ten minutes and subsequently cooled to room temperature.
Crystals were grown in a few days by the hanging drop
vapor diffusion method from a drop containing 1 mM
double-stranded chimer, 100 mM LiCl, 10 mM spermine
tetrachloride, 20 mM sodium cacodylate buffer (pH 6.0)
and 5 % (v/v) methyl-2,4-pentanediol (MPD) equilibrated against a reservoir of 1 ml of 40 % MPD at room
temperature (290 K). The X-ray diffraction data were collected from a crystal of approximate dimensions
0.25 mm 0.2 mm 0.03 mm using an R-axis IIc imaging-plate system equipped with a Rigaku rotating
anode generator using graphite monochromated CuKa
Ê ). The crystal diffracted only up to
radiation (l ˆ1.5418 A
Ê . Crystals were in orthorhombic space group I222
2.3 A
Ê , b ˆ 47.61 A
Ê and
with cell dimensions a ˆ 46.66 A
Ê . The program DENZO (Otwinovski &
c ˆ 54.05 A
Minor, 1997) was used to index the data and the data
collection details are given in Table 4.
Table 4. Crystal data and re®nement (CNS) statistics of
d(CTCCTCTTC) r(gaagagagag)
Crystal system
Space group
Cell parameters
Ê)
a (A
Ê)
b (A
Ê)
c (A
Ê 3)
Volume/base-pair (A
Ê)
Resolution (A
Ê)
Number of unique reflections (10-2.3 A
Ê)
No. reflections used (10-2.3 A
Data completeness (%)
Ê)
In the outer shell (%) (2.38-2.30 A
Rsym (%) on intensity
R-factor (%)
Rfree (%)
rmsd from ideal geometrya
Ê)
Bond lengths (A
Bond angles (deg.)
Improper angles (deg.)
a
Orthorhombic
I222
46.66
47.61
54.05
1578
2.3
2578
2486
91.4
71.3
6.9
19.9
22.2
0.007
1.2
1.4
Calculated using the parameter ®le dna-rna rep.param
Structure solution and refinement
The structure was solved by using the program
Ê 3 volume per baseAMoRe (Navaza, 1994). The 1580 A
pair clearly indicated that there was one duplex in the
asymmetric unit. The atomic coordinates of the DNARNA hybrid (Xiong & Sundaralingam, 2000) were used
as the search model. The rotation and translation search
gave a clear solution with an R-value of 41 %. The structure was re®ned by the Powell conjugate gradient energy
minimization method using the program X-PLOR
(Brunger, 1994). The DNA-RNA parameter ®le
(Parkinson et al., 1996) was used for the re®nement:
8.4 % of the re¯ections were used for the calculation of
Rfree (Brunger, 1992). Rigid body and positional re®nement brought the R-factor to 29 % and Rfree to 33.1 % for
Ê data. Both Fo ÿ Fc and 2Fo ÿ Fc maps
the 10-2.3 A
showed the looped-out adenine bulge. Further re®nement of positional and temperature parameters reduced
the R-factor and Rfree to 24.5 % and 30.2 %, respectively.
Then, simulated annealing was performed by heating
the system to 3000 K and slow cooling to 293 K in steps
of 0.5 fs. The R-factor and Rfree values at this stage were
23.6 % and 29.9 %, respectively. Inclusion of 19 water
molecules in the re®nement lowered the R-factor and
Rfree values to 20.6 % and 27.7 %, respectively, for 2486
Ê.
re¯ections with F > 2sF in the resolution range 10-2.3 A
Later, using the same number of re¯ections, the structure
was re®ned using CNS (Brunger et al., 1998). The bulk
solvent correction and cross-validated maximum likelihood approach incorporated in the simulated annealing
re®nement (Adams et al., 1997) reduced the R and Rfree
to 19.9 % and 22.2 %, respectively. Four additional water
molecules (total 23) were also located and included in
the re®nement. The CNS re®ned coordinates are used for
discussion here. The re®nement parameters are given in
Table 4.
Atomic coordinates
The atomic coordinates and the structure factors have
been deposited in the Nucleic Acid Database (Berman
et al., 1992) with accession code AH0010.
Acknowledgments
We acknowledge the support of this work by the
National Institute of Health grant GM-17378 and the
Board of Regents of Ohio for an Ohio Eminent Scholar
Chair and Endowment to M.S. We also acknowledge the
Hays Consortium Investment Fund by the Regions of
Ohio for partial support for purchasing the R-axis IIc
imaging plate.
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Edited by I. Tinoco
(Received 27 January 2000; received in revised form 28 March 2000; accepted 28 March 2000)