ABSTRACT
In order to further understand the structural role of the modified nucleoside
dihydrouridine in RNA the solution conformations of Dp and ApDpA were analyzed
by one- and two-dimensional proton NMR spectroscopy and compared with those of the
related uridine-containing compounds.
The analyses indicate that dihydrouridine significantly destabilizes the C3
'
-endo sugar conformation associated with base stacked, ordered, A-type helical RNA.
Equilibrium constants (
K
eq = [C2
'
-endo]/ [C3
'
-endo]) for C2
'
-endo-C3
'
-endo interconversion at 25
o
C for Dp, the 5
'
-terminal A of ApDpA and D in ApDpA are 2.08, 1.35 and 10.8 respectively.
Stabilization of the C2
'
-endo form was shown to be enhanced at low temperature, indicating that C2
'
-endo is the thermodynamically favored conformation for dihydrouridine.
[Delta]
H
values show that for Dp the C2
'
-endo sugar conformation is stabilized by 1.5 kcal/mol compared with Up.
This effect is amplified for D in the oligonucleotide ApDpA and propagated to the 5
'
-neighboring A, with stabilization of the C2
'
-endo form by 5.3 kcal/mol for D and 3.6 kcal/mol for the 5
'
-terminal A.
Post-transcriptional formation of dihydrouridine therefore represents a biological strategy opposite in effect to ribose methylation,
2-thiolation or pseudouridylation, all of which enhance regional stability
through stabilization of the C3
'
-endo conformer.
Dihydrouridine effectively promotes the C2
'
-endo sugar conformation, allowing for greater conformational flexibility
and dynamic motion in regions of RNA where tertiary interactions and loop
formation must be simultaneously accomodated.
Dihydrouridine is the single most common form of post-transcriptional modification in tRNA from bacteria and eukaryotes (
1
,
2
) out of >70 modifications known to occur in these two phylogenetic domains (
3
). Some of the clearest and most compelling evidence for functional roles of
tRNA modification (
4
) has been from studies which have demonstrated enhanced structural
stabilization. Examples are the stabilizing effects of ribose methylation at O-2' or thiolation of uridine at C-2 (
5
,
6
) and the influence of modification on
T
m
(
7
,
8
) and on enforcement of correct codon recognition (
9
). In contrast, much less attention has been paid to modifications that
potentially
decrease
regional stability and promote conformational flexibility of individual
nucleotide residues, properties which would be useful to accomodate loop
structure or to promote dynamic motion.
Dihydrouridine is thought to fall into this category, primarily because the non-planar base that results from the absence of a C5=C6 double bond resists
stacking (
10
,
11
). The structural properties of dihydrouridine have been studied in the
nucleoside alone, by NMR (
12
) and X-ray crystallography (
10
,
11
), and in the D loop of intact tRNAs (
13
-
15
). Although dihydrouridine-containing oligonucleotides have recently been synthesized and their
1
H NMR resonances assigned (
16
), no thermodynamic conformational analyses have been carried out in solution on dihydrouridine
monophosphate or on any dihydrouridine-containing oligonucleotides. While monophosphates clearly represent a more realistic
model than the nucleoside alone (
5
,
17
), the trinucleotide structure provides a minimal mimic of polynucleotide
structure (
18
). The sequence 2448-ApDpA-2450 has recently been found to occur in the central loop of domain
V in
Escherichia coli
23S rRNA (
19
).
In the present study, to better understand the role which dihydrouridine plays
in promoting structural flexibility in RNA, the conformational properties of Dp
and ApDpA, compared with Up and ApUpA, were analyzed in solution by one- and two-dimensional NMR spectroscopy.
Uridine and uridine 3'-monophosphate were purchased from Sigma Chemical Co. (St Louis,
MO). Dihydrouridine and dihydrouridine 3'-monophosphate were each synthesized in 98% yield by hydrogenation
at atmospheric pressure of uridine or uridine 3'-monophosphate using 5% rhodium on alumina catalyst in aqueous media
(
20
).
1
H NMR (500 MHz, D
2
O) for dihydrouridine 3'-monophosphate at 20oC, chemical shift (integrated intensity, multiplet structure,
3
J
H-H
, assignment): 5.65 p.p.m. (1, d, J
H1'-H2'
= 6.19 Hz, H1'), 4.23 (1, ddd, J
H2'-H3'
= 5.61, J
H3'-H4'
= 3.80, J
H3'-31P
= 7.85, H3'), 4.11 (1, dd, J
H1'-H2'
= 6.19, J
H2'-H3'
= 5.61, H2'), 3.93 (1, ddd, J
H3'-H4'
= 3.80, J
H4'-H5'
= 3.16, J
H4'-H5''
= 4.87, H4'), 3.60 (1, dd, J
H4'-H5'
= 3.16, J
H5'-H5''
= -12.56, H5'), 3.53 (1, dd, J
H4'-H5''
= 4.87, J
H5'-H5''
= -12.56, H5'').
The 5'-dimethoxytritylation of dihydrouridine was carried out according to
the method of Khorana and co-workers (
21
). Dimethoxytrityl chloride was added 233 mg at a time over a 7 h period, 1.4 g
total (4 mmol), to 984 mg (4 mmol) dihydrouridine dissolved in dry pyridine.
After an additional 2 h, methanol was added and the solution was evaporated
under reduced pressure. After twice co-evaporating with toluene to remove residual pyridine, the residue was
dissolved in CH
2
Cl
2
and extracted with 5% NaHCO
3
, followed by a saturated solution of NaCl and finally H
2
O. This solution was dried over MgSO
4
and evaporated to dryness. The resulting solid was loaded on a short column (3 * 4.5 cm) containing 20 g silica gel (60 Å, 200-400 mesh). The column was eluted with 70 ml CH
2
Cl
2
, 80 ml CHCl
3
, 50 ml CHCl
3
:MeOH (5:1) and 70 ml CHCl
3
:MeOH (5:2) successively. The eluates from the latter two solvents were combined
and evaporated. The identity of the final glassy solid (980 mg, 45.6% yield)
was verified by electrospray ionization mass spectrometry using a Sciex API
III+ mass spectrometer: MH
+
= 549, [M + Na]
+
= 571.
The 2'-
O
-silylation reaction was carried out according to the method of Ogilvie and
co-workers (
22
). 5'-
O
-Dimethoxytrityldihydrouridine (548 mg, 1 mmol) was dissolved in anhydrous
tetrahydrofuran (10 ml), followed by addition of dry pyridine (287 [mu]l, 3.7 mmol) and AgNO
3
(204 mg, 1.2 mmol). The reaction mixture was stirred for ~10 min then
t
-butyldimethylsilyl chloride (200 mg, 1.33 mmol) was added and the mixture
was stirred at room temperature for 5 h. The reaction products were filtered
into a 5% NaHCO
3
solution (20 ml) and extracted with CH
2
Cl
2
. The combined CH
2
Cl
2
layers were washed with H
2
O, dried over anhydrous MgSO
4
and evaporated to a viscous gum which was co-evaporated twice with toluene and twice from CH
2
Cl
2
to yield a pale yellow glassy solid (598 mg, 90.2% yield). TLC (CHCl
3
:MeOH 15:1) showed spots at
R
f
0.86 and 0.75 for the 2' and 3' silyl isomers respectively. The products were purified by flash
chromatography on a 4 * 12.5 cm column packed with silica gel (60 Å, 200-400 mesh, Merck grade). The products were eluted with CH
2
Cl
2
/MeOH/Et
3
N 100:4:0.03 to yield 250 mg (59.3%) of the 2'-silyl isomer. The identity of the product was verified by
electrospray mass spectrometry: [M + Na]
+
= 685.
5'-
O
-Dimethoxytrityl-2'-
O
-(
t
-butyldimethylsilyl)dihydrouridine (331 mg, 0.5 mmol) was dissolved in 1.8
ml dry CH
2
Cl
2
. Diisopropylethylamine (0.35 [mu]l, 2 mmol) was added, followed by addition of 146 [mu]l (0.65 mmol) 2-cyanoethyl
N
,
N
-diisopropylchlorophosphoramidite (
23
). After 4 h, another 146 [mu]l amidite were added. The reaction mixture was then diluted with ethyl
acetate (25 ml) and washed with a 5% NaHCO
3
solution (20 ml), saturated NaCl (2 * 20 ml) and H
2
O (20 ml) successively. The organic layer was dried over anhydrous MgSO
4
and evaporated to a viscous residue which was purified by flash chromatography
as described above. The partially purified material was chromatographed further
by thick layer TLC (20 * 20 cm 1 mm plate, CHCl
3
/MeOH 15:1). A UV absorbing band at
R
f
0.55 was scraped and eluted with CH
2
Cl
2
/MeOH (5:2) and evaporated to yield a colorless solid (280 mg, 64.7% yield). The
identity of the phosphoramidite was determined by electrospray mass
spectrometry: [M-H]
-
= 861.5. This was verified by
31
P NMR spectroscopy (acetone-d
6
), 150.09, 151.15 p.p.m.
The dihydrouridine phosphoramidite was dissolved in acetonitrile at 0.1 M concentration for oligonucleotide synthesis. RNA oligo- nucleotides were synthesized using solid phase phosphoramidite chemistry
on an Applied Biosystems 394 synthesizer using Perseptive Biosearch Expedite
phosphoramidites. The oligonucleotides were cleaved from the CPG support and
the protecting groups removed using 3:1 NH
3
:ethanol at 55oC for 3 h. The 2'-
t
-butyldimethylsilyl protecting groups were removed with triethylamine
trihydrofluoride (Aldrich, Milwaukee, WI) and the oligonucleotides purified on
a Supelcosil LC-18S column (4.6 * 250 mm) and 3 cm Brownlee Spheri-5 C
18
precolumn using a liquid chromatograph (System Gold; Beckman, Berkeley, CA)
with a waters UV monitor (model 440) for detection at 254 and 280 nm. A
gradient elution system of 0-50% 60:40 water:acetonitrile over 50 min with 25 mM TEAB, pH 6.0, was
used for purification. The purity and identity of each product was verified by
electrospray mass spectrometry: ApUpA, [M - H]
-
= 901.6; ApDpA, [M - H]
-
= 903.6.
The molecular model of ApDpA was constructed based on NMR and thermodynamic data
using the program INSIGHT II (BioSym Technologies, San Diego, CA). The sugar
conformations were constrained based on the thermodynamic values obtained from
coupling constant analysis and the glycosyl torsion angles fixed based on two-dimensional ROESY connectivities. The model was then energy minimized.
All NMR experiments were done using a Varian Unity 500 MHz NMR spectrometer and
a Nalorac Z-spec 5 mm indirect detection probe. The NMR samples contained 1 mM RNA in
0.7 ml 25 mM phosphate buffer, pH 7.0, containing 25 mM NaCl, 10 mM MgCl
2
and 0.2 mM EDTA. The samples were then prepared for NMR experiments by twice
lyophilizing from 99.96% D
2
O and finally dissolving in 99.996% D
2
O. Two-dimensional ROESY experiments (
24
) with a 300 ms mixing time were collected using a time-shared spinlock field of ~3500 Hz. The F
2
data were collected with 4096 total data points and 400 t
1
increments were zero filled to 2048 total points in F
1
. The TOCSY experiments (
25
) with a 30 ms mixing time were collected with the same sweep width, number of
data points and number of t
1
increments as used for the ROESY experiments. PECOSY (
26
) spectra were collected with Waltz-16
31
P decoupling and a decoupler field strength of ~500 Hz to minimize sample heating. The PECOSY data were collected with a
spectral width of 4000 Hz, centered at the water frequency, and 4096 data points were collected in the F
2
domain and 400 t
1
increments were zero filled to 2048 data points in F
1
. For Up and Dp the coupling constants J
H1'-H2'
, J
H2'-H3'
, J
H3'-H4'
, J
H3'-31P
, J
H4'-H5'
, J
H4'-H5''
and J
H5'-H5''
used for the conformational analyses were determined by iterative spin
simulations of the seven-spin system of ribose 3'-phosphate using the program LAOCOON-5. The calculated coupling constants and chemical
shifts were used to plot simulated spectra using VNMR spin simulation software.
For ApUpA and ApDpA NMR assignments for each residue were made on the basis of
ROESY and TOCSY experiments. The coupling constants J
H3'-H4'
were not clearly determined from one-dimensional spectra for these oligonucleotides because of spectral
overlap. It has been shown for a number of nucleotides that the value of J
H1'-H2'
+ J
H3'-H4'
is equal to 10 Hz (
27
) and this value has been used in several previous thermodynamic analyses of
oligonucleotide sugar conformation (
5
,
6
,
28
,
29
). To justify the use of this assumption in assigning values for J
H3'-H4'
in the oligonucleotides, J
H1'-H2'
+ J
H3'-H4'
values were measured over the entire temperature range for Up and Dp. The mean
value calculated was 9.9 +- 0.1 Hz. J
H3'-H4'
coupling constants for each residue in the oligonucleotides were also measured
from
31
P decoupled two-dimensional PECOSY experiments run at 5 and 25oC and verified that the sum J
H1'-H2'
+ J
H3'-H4'
was equal to ~10 Hz. We therefore assigned J
H3'-H4'
values for the oligonucleotide substituents according to the formula J
H3'-H4'
= 9.9 - J
H1'-H2'.
All vicinal proton-proton coupling constants were measured in triplicate at each temperature
(error +-<0.05 Hz) and the mean values for each used in the conformational
analyses. The determination of fractional populations of the C2'-endo and C3'-endo forms at various temperatures was performed using
a version of the PSEUROT program written in C and employing a linear least
squares optimization routine. The C version was compared with the original
PSEUROT program from Altona and co-workers (
30
) and found to give identical results for several common nucleotides. A [Delta][chi]
i
[beta]-substituent
appropriate for nucleoside 3'-monophosphates was used for the analysis. The populations of C2'-endo and C3'-endo conformers resulting from the above
PSEUROT analyses were used in individual van't Hoff plots to calculate the
average [Delta]
H
of the C3'-endo <-> C2'-endo equilibrium.
The H1' proton resonance regions of the 500 MHz
1
H NMR spectra of Up, Dp, ApUpA and ApDpA are shown in Figure
1
. The H1' proton resonances for Up and Dp were completely resolved over the entire
temperature range from 5 to 40oC, allowing straightforward measurement of J
H1'-H2'
scalar coupling constants in each case. All chemical shifts and coupling
constants for the seven-spin system of ribose in each nucleotide were iteratively calculated using
the program LAOCOON-5. Errors in the calculation of chemical shifts and coupling constants
were <0.05 Hz. The chemical shifts and coupling constants calculated for each
nucleotide were used to plot simulated spectra at each temperature. The
simulated and observed spectra of Dp at 5oC are shown in Figure
2
. Simulated spectra at every temperature were directly superimposable upon the
experimental spectra. The J
H1'-H2'
, J
H2'-H3'
and J
H3'-H4'
coupling constants calculated from simulated spectra were used in PSEUROT
analyses for determination of the pseudorotational equilibria in Up and Dp. The
H1' proton resonances for ApUpA and ApDpA were assigned on the basis of
dipolar and scalar coupling connectivities determined by ROESY and TOCSY
experiments. The H1' resonances for ApUpA were resolved over the entire temperature range and
those of ApDpA were resolved at every temperature except 20oC, where the J
H1'-H2'
coupling constants for the terminal A residues could not be determined due to
spectral overlap.
Table 1
Recent studies indicate that very accurate analyses of pseudorotational
equilibria in nucleotides can be performed using the program PSEUROT (
31
). The fractional populations of the C2'-endo and C3'-endo forms at 5 and 25oC, along with equilibrium constants ([C2'-endo]/[C3'-endo]) calculated using
the formalism introduced by de Leeuw
et al
. (
30
), are shown in Table
1
. The fractional population of the C2'-endo form is increased from 46 to 68% at 25oC upon saturation of the C5=C6 double bond of Up to form Dp.
The equilibrium constant [C2'-endo]/[C3'-endo] correspondingly increases from 0.85 to 2.08,
indicating that dihydrouridine stabilizes the C2'-endo form at the expense of the C3'-endo form. When dihydrouridine is substituted for
uridine in a small oligonucleotide the effect is of even greater magnitude. The
fractional population of the C2'-endo form for D in ApDpA is 91%, compared with 54% for U in ApUpA.
Interestingly, at lower temperatures this effect is amplified, with the
substitution of D for U causing an increase in the C2'-endo population from 54 to 96% at 5oC. The effect at lower temperature is also propagated to the 5'-neighboring A residue, which experiences an
increase in the C2'-endo conformer from 53 to 67% upon the substitution of D for U, the
equilibrium constant [C2'-endo]/[C3'-endo] being increased in the latter from 1.10 to 2.06.
To further elucidate the effect on sugar conformation upon substitution of
dihydrouridine for uridine enthalpy differences between the C2'-endo and C3'-endo forms of ribose in the related nucleotides were
calculated. Because the subject of the present study is to elucidate the role
of dihydrouridine in tRNA, where rotations around the exocyclic bonds are
restricted for both C2'-endo and C3'-endo forms, [Delta]
S
values, which depend mainly on the difference in restriction of rotations
around exocyclic bonds (
5
), are not considered here. Figure
3
shows the temperature dependence of the equilibrium constants [C2'-endo]/ [C3'-endo] for Up, Dp and the individual residues of ApUpA
and ApDpA. [Delta]
H
difference values are listed in Table
2
. A comparison of [Delta]
H
values for Up and Dp shows that saturation of the C5=C6 double bond of Up to
form Dp results in stabilization of the C2'-endo form by 1.5 kcal/mol. A comparison of [Delta]
H
values between the U residue in ApUpA and the D residue in ApDpA is even more
striking, indicating that D favors the C2'-endo sugar conformation by 5.3 kcal/mol in the oligonucleotide.
Furthermore, this effect is propagated in the oligonucleotide to the 5'-neighboring A residue, where the C2'-endo sugar conformation undergoes net stabilization of
3.6 kcal/mol upon replacement of the neighboring U with D. A similar
propagation effect is not seen for the 3'-neighboring A, which still favors the C3'-endo sugar conformation in ApDpA.
A molecular model of ApDpA based on NMR data is shown in Figure
4
and illustrates the preferred conformations of each residue as determined from
the thermodynamic and NMR data presented in this paper. It is not intended to
represent a high resolution NMR-based solution structure. The 5'-A residue and the D residue are shown in the C2'-endo sugar conformation, which are both strongly
favored according to their [Delta]
H
values. ROESY data show an intense NOE between the H8 proton of the 5'-A residue and its own H1' proton (data not shown) and no NOE cross-peaks to other sugar protons, indicating that the base
of the 5'-A adopts a largely
syn
glycosyl torsion angle, as shown. A reviewer has pointed out the likelihood
that a hydrogen bond between the 5'-OH and N3 of adenine, as established by X-ray crystallography (
32
), may influence the sugar conformation of the 5'-A. This possibility is recognized, but no NMR data are available to
address this point. No NOEs are observed between the 5'-A and the following D nucleoside and the H6 protons of D only give
NOEs to the D sugar H2' and H3' protons and no sequential NOEs to the 5'-A. The 3'-A residue is shown adopting the C3'-endo sugar conformation, in
accord with the enthalpy data and supported by sequential NOE cross-peaks from the H8 proton to H2' and H3' of D, as well as to its own sugar protons.
Dihydrouridine is one of the most highly conserved modified nucleosides in
transfer RNA, occuring primarily at positions 16, 17, 20, 20a and 20b in loop I
(D loop) and occasionally position 47 in the variable loop of the cloverleaf
structure of tRNA (
1
,
2
). The main structural features of dihydrouridine as established by X-ray crystallographic structures of dihydrouridine and dihydrouridine 3'-monophosphate (
11
,
33
) are 2-fold: puckering of the pyrimidine ring with C6 0.47 Å out of the plane described by the remaining base atoms; adoption
of the C2'-endo sugar conformation by the ribose moiety. The latter finding is
corroborated by crystal structures of tRNA
Phe
and tRNA
Asp
(
15
,
34
), which show that dihydrouridine and several neighboring nucleosides adopt the
C2'-endo sugar conformation. Conformational analyses of dihydrouridine
in solution, however, have been limited to the nucleoside (
12
), which show the ribose moiety slightly prefers the C2'-endo sugar conformation, although rapid interconversion between
ring-puckered conformers occurs.
In the present study, thermodynamic parameters for the principal ribose
conformers of dihydrouridine monophosphate and a dihydrouridine-containing oligonucleotide, ApDpA, have been determined in solution, to
provide further insights into the functional role of D.
The conformational studies (including calculation of [Delta]
H
values) of Up, Dp, ApUpA and ApDpA presented here show that each residue of
ApUpA is in rapid equilibrium between the C2'-endo and C3'-endo sugar conformations with [C2'-endo] = 52-54% at 5oC and 53-55% at 25oC, a finding
which agrees well with data for the majority of trinucleoside diphosphates
containing uridine studied by Lee and Tinoco (
18
). However, the C3'-endo sugar conformation of Up is significantly destabilized as a
result of the saturation of its C5=C6 double bond to form Dp. It is known that
substituents at C5 of uridine which strengthen the bonding interaction between
a lone pair of electrons at O
4
' and the [pi]
*
orbital of the C5=C6 double bond cause uridine to favor small [chi] angles and increase populations of the C3'-endo conformers (
35
,
36
). The absence of a C5=C6 double bond in dihydrouridine would disfavor this
interaction, thereby disfavoring small [chi] angles and decreasing the population of C3'-endo conformers. In addition, destabilization of the C3'-endo conformation is probably enhanced through
steric interaction between the methylene protons of C6 with the ribose moiety (
33
). The destabilization of the C3'-endo form is further enhanced when D is substituted for U in a
small oligonucleotide. The presence of dihydrouridine in this case has three
distinct effects on RNA structure. The first is to promote the C2'-endo sugar conformation (destabilizing the C3'-endo form) for the D nucleoside compared with the U
that it replaces. The second is to cause complete destacking of the bases in
the oligonucleotide. The third is that the 5'-neighboring nucleoside (in this case A) experiences a concomitant
destabilization of the C3'-endo sugar conformation, favoring C2'-endo. The C2'-endo conformer is inherently more
flexible (
37
), accomodating a wider range of glycosyl torsion angles (
38
). The fact that destabilization of the C3'-endo form occurs in the mononucleotide as well as the
oligonucleotide indicates that destabilization of the C3'-endo form in ApDpA is not solely due to absence of stacking due to
non-planarity of the D heterocycle. Although there is a relationship between
adoption of the C2'-endo sugar conformation by the first two nucleotides of ApDpA and
destacking of the bases in the trinucleotide, any conclusions regarding cause
and effect in this inter-relationship would be unwarranted without study of a wider variety of
dihydrouridine-containing oligonucleotides as sequence models.
The role of several post-transcriptionally modified nucleosides in stabilizing RNA structure has
been previously studied using NMR techniques. Sugar methylation is the most
well-characterized example, where 2'-
O
-methylation stabilizes the C3'-endo sugar conformation of Up by 0.8 kcal/mol (
5
) and also results in an increase in duplex stability for oligonucleotides (
39
). Other modifications which stabilize RNA are 2-thiolation of ribosylthymine, which stabilizes the C3'-endo sugar conformation by 1 kcal/mol (
28
),
N
4
-acetylation of cytidine, which stabilizes the C3'-endo sugar conformation by 1.22 kcal/mol, and
N
4
-acetylation of cytidine in combination with 2'-
O
-methylation, which results in C3'-endo stabilization by 1.53 kcal/mol (
29
). The role of post-transcriptional modifications such as these in nature can be seen in the
example of tRNAs from archaeal hyperthermophiles, where the extent of post-transcriptional modification correlates with cell growth temperature and
leads to a net contribution to
T
m
of ~20oC (
8
). Pseudouridylation is another modification which is a complementary strategy
for stabilization of local RNA structure (
40
,
41
).
In contrast, results of the present conformational analysis of dihydrouridine
indicate that its structural role is opposite to that of the nucleosides listed
above. Whereas the latter modifications
stabilize
the C3'-endo sugar conformation of RNA by anywhere from 0.8 to 1.5 kcal/mol
(
5
,
29
), dihydrouridine
destabilizes
the C3'-endo sugar conformation with equal magnitude at the mononucleotide
level (1.5 kcal/mol) and at a much greater magnitude at the oligonucleotide
level (5.3 kcal/mol).
The refined crystal structure of tRNA
Phe
(
15
) shows that the 12 residues that adopt the C2'-endo sugar conformation are all in single-stranded loops and regions where stretching of the
polynucleotide backbone is required. Furthermore, from a stereo drawing
representation that shows sites of the C2'-endo residues (see supplementary material in ref.
15
) it is striking that all of these residues cluster in the corner of the
molecule that has the highest concentration of tertiary interactions. Of all
the residues adopting the C2'-endo sugar conformation in tRNA
Phe
and tRNA
Asp
the majority are in or near the dihydrouridine loop (seven of 12 residues in
tRNA
Phe
and seven of 10 in tRNA
Asp
) (
15
,
34
). It is apparent, at least for tRNA
Asp
, that the residues in the variable loop and T[psi]C loop which adopt the C2'-endo form (including A9, U48 and A58) belong to a region of
the polynucleotide chain where intercalation of bases occurs (
34
), which is facilitated by the additional flexibility of the C2'-endo nucleosides.
All of this suggests that nucleosides in the C2'-endo sugar conformation are required in the D loop and to some
extent the variable loop and T[psi]C loop in order to facilitate tertiary interactions, while at the same time
lowering the energy for loop formation in the same region of the molecule. The
results of the present study, together with X-ray crystallographic data (
10
,
11
,
15
,
33
,
34
), indicate that the responsibility for promoting the C2'-endo sugar conformation in loop I of transfer RNA probably falls
heavily upon dihydrouridine. The recent discovery of dihydrouridine in the
central loop of domain V in 23S rRNA from
E.coli
(
19
) suggests that this role for dihydrouridine might be more universal, and not merely limited to tRNA. Further, in organisms that
grow optimally at low temperatures (psychrophiles), where conformational
flexibility of RNA must be preserved, exceptionally high levels of
dihydrouridine have been found (J. J. Dalluge and J. A. McCloskey, unpublished
observations).
The authors wish to thank Jay Olsen for his technical assistance with all NMR
experiments and Richard H. Griffey (ISIS Pharmaceuticals) for providing his
PSEUROT C program. This work was supported by NIH grant GM29812 to J.A.M. and
NSF grant MCB-9317196 to D.R.D. The NMR facility is supported by NIH grants P30 CA42014
and S10 RR06262.
Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84112,
USA
C2'-endo
C3'-endo
[C2'-endo]/[C3'-endo]
b
5oC
25oC
5oC
25oC
5oC
25oC
45
46
55
54
0.82 (0.01)
0.85 (0.02)
72
68
28
32
2.51 (0.02)
2.08 (0.02)
53
55
47
45
1.10 (0.01)
1.19 (0.03)
A
54
54
46
46
1.17 (0.02)
1.17 (0.01)
AU
52
53
48
47
1.08 (0.02)
1.13 (0.03)
67
58
33
42
2.06 (0.03)
1.35 (0.02)
A
96
91
4
9
22.25 (0.02)
10.76 (0.02)
AD
53
55
47
45
1.10 (0.01)
1.25 (0.03)
REFERENCES
Return