(Received for publication, March 14, 1995)
From the
Knowledge of the sites, structures, and functional roles of
posttranscriptional modification in rRNAs is limited, despite steadily
accumulating evidence that rRNA plays a direct role in the peptidyl
transferase reaction and that modified nucleotides are concentrated at
the functional center of the ribosome. Using methods based on mass
spectrometry, modifications have been mapped in Escherichia coli 23 S rRNA in the central loop of domain V, a region of established
interaction between 23 S RNA and tRNA. Two segments of RNA were
isolated following protection with oligodeoxynucleotides and nuclease
digestion: residues 2423-2473 (51-mer) and 2481-2519
(39-mer). Dihydrouridine was located at position 2449, within the RNase
T There is substantial biochemical evidence that implicates 23 S
ribosomal RNA in the translational process (reviewed in (1) ).
In particular, the highly conserved central loop of domain V (residues
2043-2625(2, 3) ) has been shown to be a
fundamental component of the peptidyl transferase activity. The
evidence is principally based on chemical footprinting experiments (4) and photoaffinity cross-linking studies (reviewed in (5) ) using components of the translational apparatus (e.g. tRNA, mRNA, ribosomal proteins) or inhibitors of ribosomal
function such as antibiotics (reviewed in (6) ). The recent
report that the peptidyl transferase function of Thermus aquaticus ribosomes is retained by the 23 S rRNA after extensive depletion
of ribosomal proteins (7) demonstrates that 23 S rRNA plays a
direct and major role in the peptidyl transferase reaction. Woese (8) has defined three criteria for functionally interactive
regions of ribosomal RNA: 1) universal sequence conservation, 2) single
strandedness (defined by secondary structure), and 3) surface proximity
(defined by three-dimensional structure). Further, it has been
suggested that the occurrence of posttranscriptional modifications in
functionally interactive regions is evidence that these modifications
are involved in modulation of specific structural features(9) .
The primary structure of the central loop of domain V of 23 S rRNA
contains nucleotide sequences that are almost universally
conserved(10) , including several nucleotides that are
posttranscriptionally modified (11, 12, 13, 14, 15) .
However, in contrast to tRNA, for which the chemical
identities(16) , sequence locations(17) , and
functional roles(18, 19) of modification have been
studied in greater detail, the structures and functions of modification
in rRNA have received considerably less attention, and detailed
examination has been confined to relatively few organisms, principally
higher eukaryotes (20) and Escherichia coli. In the
case of E. coli 23 S rRNA, approximately 22 sites of
modification are reported (summarized in (14) and (15) ), but some assignments are regarded as tentative, and a
total census of modifications is yet incomplete. Because of the
functional importance of domain V in 23 S rRNA, we have examined
modifications in two segments around the central loop, residues
2423-2473 and 2481-2519, using a combination of methods
based on mass spectrometry designed for characterization and sequence
placement of modified nucleotides(21) . We recently reported
the detection of the modified nucleoside 5,6-dihydrouridine in a total
nucleoside digest of E. coli 23 S rRNA(22) , an
observation in concert with an early literature report(23) . We
presently report that dihydrouridine occurs at position 2449, close to
the CCA-aminoacyl terminus of tRNA in the ribosomal P-site established
by cross-linking experiments(24) .
Figure 3:
Electrospray mass spectrum of
oligoribonucleotide 7-mer produced from RNase T
The sequence location and structural characterization of
posttranscriptionally modified residues in two segments (totaling 90
nucleotides) of the primary sequence of E. coli 23 S rRNA
(2904 nucleotides) as described in the following sections is based on a
nuclease protection strategy used in combination with recently
developed mass spectrometric methodologies(21) , in which
posttranscriptional modifications are detected by mass shifts (e.g. U
Figure 1:
LC/MS analysis of nucleosides from an
enzymatic digest of 0.1 nmol of E. coli 23 S rRNA. Panel
A, reversed phase HPLC separation, with detection by UV absorbance
at 254 nm. Peak identities are: 1, dihydrouridine (D); 2, pseudouridine (
The
presence of a previously unidentified ribonucleoside species of m/z 259 is detected at 10.9 min (Fig. 1C). A protonated molecular mass of 259 Da is
consistent with a monomethyl uridine derivative, M The detection of
2-methyladenosine and N
Figure 2:
LC/MS analysis of nucleosides from an
enzymatic digest of 0.9 nmol of the 51-mer fragment (residues
2423-2473) from E. coli 23 S rRNA. Panel A,
reversed phase HPLC separation, with UV detection at 254 nm. (D,A)
denotes a dinucleotide containing D and A. Panel B, mass
spectrum recorded at 3.3 min showing MH
Inspection
of the E. coli 23 S rDNA sequence shows that the unmodified
base composition of the 51-mer is
C
Figure 4:
LC/MS analysis of nucleosides from an
enzymatic digest of 0.15 nmol of the oligoribonucleotide 7-mer
(residues 2448-2454) from E. coli 23 S rRNA. Panel
A, reversed phase HPLC separation, with UV detection at 254 nm.
(D,A) denotes a dinucleotide containing D and A. Panel B, mass
spectrum recorded at 18.9 min, showing ions from constituents A (m/z 136, BH
Figure 5:
LC/MS analysis of nucleosides from an
enzymatic digest of 0.9 nmol of the 39-mer fragment (residues
2481-2519) from E. coli 23 S rRNA. Panel A,
reversed phase HPLC separation, with UV detection at 254 nm. Panels
B-D, reconstructed ion chromatograms for m/z 245,
258, and 282, respectively.
Figure 6:
HPLC analysis of an enzymatic digest of
oligonucleotide M
Figure 7:
HPLC analysis of an enzymatic digest of
oligonucleotide M
Extensive evidence from studies of tRNA implies that
posttranscriptional modifications in all RNAs are generally highly
conserved (e.g.(17) ) and play functional roles in
(among others, (18) ) modulation of tertiary structure (e.g.(36) and (37) ). However, much less is
known about the chemical identities and sites of posttranscriptional
nucleotide modification in ribosomal RNA. In particular, 23 S ribosomal
RNA has become a central model for understanding the functional role of
rRNA in the peptidyl transferase reaction(1) , but current
knowledge of posttranscriptional modification in 23 S rRNA is both
incomplete and in some cases
contradictory(12, 13, 14, 15, 30) .
Virtually nothing is presently known about specific functional roles of
these modifications, but their generic importance is implied by the
striking correlation that nearly all of the known sites of modification
lie within or close to the functional center of the
ribosome(5) . Ultimately, these modifications must be
reconciled with the tertiary structure and dynamics of 23 S rRNA
participation in translation. To some extent, past difficulties in
making correct assignments of modification in rRNA can be attributed to
use of insufficiently rigorous methods of characterization (discussed
in (16) ) coupled with the large size of the RNA. In the
present work, these problems have been reduced by the use of mass
spectrometry-based methods(21, 38) . In particular,
measurement of the intrinsic molecular property of mass, when made in
conjunction with HPLC in the LC/MS experiment, provides a considerably
more objective means for nucleoside identification compared to reliance
solely on chromatographic mobility. The problem of the size of 23 S
rRNA (2904 nucleotides) proved to be significantly more important than
in the case of 16 S rRNA (1542 nucleotides) when studied using the same
methods(21) . We calculated that complete RNase T We have directed our attention principally to modification sites in
the central loop of domain V of 23 S rRNA, which constitutes (a portion
of) the catalytic center of the ribosome as defined by the multiplicity
of sites of interaction between 23 S rRNA and other components of the
translational apparatus (e.g. the aminoacyl termini of tRNAs
in both the ribosomal A- and P-sites)(1) . Residues
2423-2473 were selected for analysis because of the existence of
an unidentified uridine derivative at position 2449 and the presence of
two other ``known'' posttranscriptional modifications
(m In the present study,
six sites of posttranscriptional modification were determined by mass
spectrometry in the two segments of domain V that were studied:
m Dihydrouridine-2449 in the central loop of domain V, one of the most
highly conserved segments of rRNA, constitutes the first sequence
placement of D outside of tRNA(16) . The detection of D (Fig. 2B) confirms our initial observation (22) and the early report of Johnson and Horowitz (23) (the latter work, in which identification was based on
two-dimensional chromatography, also cites D in E. coli 16 S
rRNA, but this finding is discounted in view of more recent
studies(21) ). The initial discovery of modification at
position 2449 in E. coli 23 S rRNA stems from early work on
oligonucleotide fingerprinting, applied in pursuit of the total primary
sequence (e.g.(39) and (40) ). At that time,
before the gene sequence was available, individual oligonucleotides
were sequenced, but their correct positions in the overall RNA
structure were usually not known. Hydrolysis of 23 S RNA by RNase
T The failure to establish modification at C-2501 was unexpected.
Previously reported data showed a strong reverse transcription stop,
which was interpreted to indicate modification at this
site(15) . In our hands, isolation of the RNase T The finding
of dihydrouridine in the central loop of domain V (Fig. 8), a
region that is viewed as conserved and highly compact(5) ,
raises questions of function and whether this specific modification is
found at the same position in other 23 S rRNAs. It is plausible to
assume that D-2449 serves to enforce conformational flexibility in this
vicinity of the loop to accommodate dynamic motion during translation.
This interpretation is supported by the structural properties of
dihydrouridine, earlier studied alone and in the D-loop of bacterial
and eukaryotic tRNAs. Saturation of the uridine C5=C6 double
bond results in a base moiety that is non-planar with C6 0.466 Å
out of the plane of the base(41, 42) . The molecule
therefore resists base stacking, as corroborated by x-ray
crystallographic structures of tRNA
Figure 8:
Secondary structure model of the highly
conserved central loop of domain V, showing known sites of
posttranscriptional modification. The locations and identities of other
reported sites of modification are: m
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
hydrolysis product 2448-ADAACAGp-2454, as evidenced by a
molecular mass 2 daltons higher than the gene sequence-predicted mass.
This nucleoside, which is nearly ubiquitous in tRNA (where it is
involved in maintenance of loop structure), is two bases from A-2551, a
previously determined site of interaction between 23 S RNA and the
CCA-aminoacyl terminus of tRNA at the ribosomal P-site. The
oligonucleotide 2496-CACmCUCGp-2502 was isolated and accurately mass
measured, and its nucleoside constituents were characterized by high
performance liquid chromatography-mass spectrometry; there was no
evidence of modification at position 2501 as implied by earlier work.
Using similar techniques, the modified adenosine at position 2503 was
unambiguously determined to be 2-methyladenosine in the fragment
2503-m
A
Gp-2505.
Isolation of rRNA
50 S ribosomal
subunits were prepared from frozen E. coli MRE 600 cells (1/2
log phase; Grain Processing Corp., Muscatine, IA)(25) .
Ribosomal RNA (23 and 5 S) was isolated from 50 S subunits by
extraction with phenol and chloroform(25) . 23 S rRNA was
separated from 5 S rRNA by precipitation of the former in the presence
of 2 M LiCl. Purity of the 23 S rRNA isolate was assessed by
gel electrophoresis (1% agarose; 3.5 50-mm tube) using an
Applied Biosystems model 230A micro-preparative electrophoresis system.
Synthesis of Complementary DNA
DNA
oligonucleotides complementary to the gene sequence of E. coli 23 S rDNA from positions 2423 through 2473 (51-mer) and from
positions 2481 through 2519 (39-mer), respectively, were synthesized on
a 1.0-µmol scale using an Applied Biosystems model 380B DNA
synthesizer at the University of Utah Protein/DNA Core Facility. The
DNA 51-mer and 39-mer were purified by DEAE anion exchange HPLC,(
)as described below.
Preparation of Specific rRNA Fragments by Nuclease
Protection of an RNA:DNA Heteroduplex
In separate
preparations, synthetic oligodeoxynucleotides (100 nmol), complementary
to residues 2423-2473 and 2481-2519, respectively, of E. coli 23 S rRNA were combined with E. coli 23 S
rRNA (10 nmol) in annealing buffer (100 mM KCl, 10 mM NHOAc, pH 5.3, 10 mM Zn(OAc)
, 1
mM 2-mercaptoethanol, 5% glycerol). The RNA was denatured by
heating at 100 °C for 2 min and then quickly cooled in an ice-water
slush. The oligodeoxynucleotide was annealed to the ribosomal RNA
during a 3-h incubation at 37 °C. The RNA:DNA hybrid was then
treated with mung bean nuclease (10,000 units; Life Technologies, Inc.)
at 37 °C for 30 min to hydrolyze all RNA not protected in the
RNA:DNA duplex. The enzyme was removed by extraction with phenol and
chloroform, and the RNA:DNA duplex was precipitated with 2 volumes of
ethanol, pelleted by centrifugation, and dried in a Savant SpeedVac
vacuum centrifuge. The sample was redissolved in 10 mM NH
OAc, pH 5.3, and 5 mM Mg(OAc)
and digested with DNase I (1,000 units; Life Technologies, Inc.)
at 37 °C for 30 min. The rRNA 51-mer(2423-2473) and
39-mer(2481-2519) were separated from the resultant mixture of
small oligodeoxynucleotides (n
6) by DEAE-anion exchange
HPLC, as described below.
Enzymatic Hydrolysis of rRNA
E. coli 23 S rRNA (0.1 nmol) and enzymatically produced fragments thereof
(0.9 nmol of 51-mer and 0.15 nmol of a 7-mer prepared from the 51-mer;
0.8 nmol of 39-mer and 0.16 nmol of a trimer and a 7-mer prepared from
the 39-mer) were hydrolyzed to nucleosides using nuclease P1 (Life
Technologies, Inc.), venom phosphodiesterase (Sigma), and alkaline
phosphatase (Calbiochem)(26) . The RNA 51-mer (8.0 nmol) and
39-mer (8.0 nmol) from E. coli 23 S rRNA were digested using
RNase T (2,000 units; Ambion) for 30 min at 37
°C(21) .
High Performance Liquid
Chromatography
The synthetic DNA 51-mer and 39-mer were
purified by anion exchange chromatography on a Nucleogen DEAE
60-7 column (4 125 mm) and precolumn (4
30 mm)
(The Nest Group, Southborough, MA) installed on a Beckman model 332
liquid chromatograph with a Beckman model 160 UV monitor for detection
at 254 nm. The eluant was a linear gradient of 0.025 M triethylammonium bicarbonate, pH 6.4, 20% CH
CN to 1.0 M triethylammonium bicarbonate, pH 7.8, 20% CH
CN
at 1.0 ml/min for 50 min. The RNA 51-mer and 39-mer were separated from
mixtures of oligodeoxynucleotides by DEAE-anion exchange chromatography
using the same conditions described above. The oligonucleotide mixtures
from complete RNase T
digestion of the E. coli 23
S RNA 51-mer and 39-mer were fractionated by anion exchange
chromatography(21) . Subsequent reversed phase purification and
subfractionation of specific DEAE oligonucleotide fractions was
accomplished by HPLC with a Supelcosil LC-18S column (4.6
250
mm) and precolumn (4.6
30 mm) using a linear gradient of 0.025 M triethylammonium bicarbonate, pH 6.4, 2% CH
CN
and CH
CN:H
O (4:6)(21) .
Directly Combined High Performance Liquid
Chromatography-Mass Spectrometry
The mass spectrometer for
LC/MS analyses consists of a non-commercial quadrupole mass analyzer,
with thermospray ionization interface (Vestec Corp, Houston, TX),
controlled by a Vector/One data system (Teknivent Corp., St. Louis,
MO). Separation of nucleosides was carried out with a Beckman 322
liquid chromatograph with Waters 440 dual wavelength (254 and 280 nm)
UV monitor placed in series between the chromatograph and mass
spectrometer, using a Supelcosil LC-18S 4.6 250-mm column and
4.6
30-mm C
precolumn (Supelco, Bellefonte, PA)
thermostatted at 32 °C. The HPLC gradient elution system of Buck et al.(27) with 0.25 M ammonium acetate (pH
6.0) and acetonitrile was used with minor modifications in the gradient
profile (28) . Mass spectra were acquired every 1.7 s during
the 35-min chromatographic separation. The instrument, procedures, and
interpretation of data for characterization of nucleosides in RNA
hydrolysates have been described(28, 29) .
Electrospray Mass Spectrometry of
Oligonucleotides
Oligonucleotides from RNase T hydrolysis of the 51-mer and 39-mer fragments from E. coli 23 S rRNA were dissolved in H
O and diluted with
CH
OH to 1:9 (v/v) at a final sample concentration of 8.0
pmol/µl (assuming quantitative recovery from two chromatographies).
Samples were continuously infused at a flow rate of 2.0 µl/min
using a Harvard model 22 syringe pump into the electrospray ion source
of a Sciex API III+ mass spectrometer (Ontario), operated with
quadrupole-3 in rf-only mode. The mass spectrum presented in Fig. 3is the sum of ten scans (m/z 200 through m/z 1200) acquired at a rate of 40 s/scan.
Calibration of the mass scale was accomplished using synthetic
oligodeoxynucleotides as mass reference standards(21) .
hydrolysis
of the 51-mer (residues 2423-2473) from E. coli 23 S
rRNA. Experimentally measured m/z values and
resulting base compositions are listed in Table 1.
D, +2 Da) not predicted by the rDNA sequence.
Total Nucleoside Analysis of E. coli 23 S
rRNA
A UV absorbance chromatogram corresponding to the
LC/MS analysis of ribonucleosides present in an enzymatic hydrolysate
of 0.1 nmol of intact E. coli 23 S rRNA is shown in Fig. 1A. Reconstructed ion chromatograms based on
specific mass values characteristic of nucleosides of interest are
presented in Fig. 1, B-D. In Fig. 1B, the ion of m/z 247, at 3.3
min elution time(28) , corresponds to the protonated molecular
ion (MH) of dihydrouridine. The other m/z 247 signals at 5.1 and 7.1 min result from minor isotope peaks of
cytidine and uridine, respectively, which are present in great molar
excess relative to D (gene sequence indicates the RNA composition
C639U591G912A762). The total number of D residues in E. coli 23 S rRNA is presently not known but is presumed to be one.
); 3, unknown (U*); 4, 5-methylcytidine; 5, 2`-O-methylcytidine
(Cm); 6, 5-methyluridine (m
U); 7,
7-methylguanosine (m
G); 8,
2`-O-methyluridine (Um); 9, adenosine 2`,3`-cyclic
phosphate (A>p); 10, 1-methylguanosine; 11,
2`-O-methylguanosine; 12, N
-methylguanosine (m
G); 13,
dinucleotide of component 3 and A; 14, 2-methyladenosine
(m
A); 15, N
-methyladenosine
(m
A). The major ribonucleosides are lettered.
Shoulders on the rightsides of C, U, G, and A peaks
are characteristic chromatographic artifacts resulting from injection
of large quantities of the four major nucleosides. Triangles denote solvent impurities. Panels B-D,
reconstructed ion chromatograms for m/z 247, 259, and
282, respectively. See text for discussion of
assignments.
258. Based on relative retention time(28) , the known (16) RNA methyluridine isomers 3- and 5-methyluridine,
1-methylpseudouridine, and 2`-O-methyluridine are excluded.
Structural characterization and sequence location of the M
258 nucleoside, which is located outside of
domain V, is being investigated separately. Also observed are ions of m/z 259 at 14.8 and 16.7 min that correspond to the
protonated molecular ions of 5-methyluridine (m
U) and
2`-O-methyluridine, respectively. LC/MS analysis of
2`-O-methylated nucleosides (and their 3` neighbors), prepared
from RNase T
hydrolysis of E. coli 23 S rRNA,
shows an ion of m/z 113 at 16.7 min (data not shown)
that corresponds to the protonated non-methylated base ion
(BH
) of uracil. Clearly, these results do
not support the earlier report of 3-methyluridine based on HPLC data
alone (30) but agree with early reports of
2`-O-methyluridine in E. coli 23 S rRNA (e.g.(31) ). The minor ion profiles of m/z 259, at 11.7 and 13.5 min in Fig. 1C, represent
the
C isotope peaks of 5-methylcytidine and
2`-O-methylcytidine, respectively.
-methyladenosine is shown
in Fig. 1D. The thermospray mass spectra of these
isomers are similar; both species produce a protonated molecular ion of m/z 282 (corresponding to M
281)
at 24.8 min (m
A) and 25.3 min (m
A) and
protonated base fragment ion of m/z 150 (data not
shown). These isomers are differentiated by their relative retention
times (28) and their characteristic UV absorbance ratios (A
/A
) (data not shown).
This result agrees with an earlier report based on HPLC data (30) that both m
A and m
A are present in E. coli 23 S rRNA. Determination of the sequence location of
m
A is presented below. N
,N
-Dimethyladenosine was
previously reported in substoichiometric amount in E. coli 23
S RNA (30) but is clearly absent in the present study (t
30.6 min; region not shown in Fig. 1A). Detection in the earlier work is judged to
result from contamination by 16 S RNA.
Total Nucleoside Analysis of Nucleotide Region
2423- 2473
An RNA 51-mer was prepared by
hybridization of an oligodeoxynucleotide (complementary to positions
2423-2473) to 23 S rRNA, followed by enzymatic removal of the
non-hybridized RNA and excess cDNA. This approach is conceptually
similar to the strategy of Maden(32) , although we have made
several changes in experimental detail (see ``Materials and
Methods''). The yield of RNA 51-mer (8.9 nmol) was determined by
UV absorbance at 260 nm. An aliquot (1/10 of the sample) was
enzymatically hydrolyzed to nucleosides and analyzed by LC/MS (Fig. 2A). Mass spectra from selected time points are
presented in Fig. 2, B-D. Fig. 2B was recorded at 3.3 min and reveals ions of m/z 115 and 247 corresponding to BH and
MH
ions of dihydrouridine(29) . Fig. 2C shows the mass spectrum recorded at 3.5 min and
reveals the MH
ion of pseudouridine at m/z 245. The protonated base (aglycone) ion,
BH
of m/z 166 at 19.7
min, corresponds to N
-methylguanosine (Fig. 2D). The MH
ion, usually of low
abundance, was observed in the mass spectrum of this component in the
analysis of a separate 51-mer isolate (data not shown).
(m/z 247) and BH
(m/z 115) ions
characteristic of D. Panel C, mass spectrum recorded at 3.5
min showing MH
ion of pseudouridine (
), m/z 245. Panel D, mass spectrum recorded at 19.7 min showing
an ion of m/z 166 corresponding to the
BH
ion of N
-methylguanosine (m
G). Peaks denoted
by triangles are from background
ions.
U
A
G
. The molar
ratio of pseudouridine relative to uridine was determined to be 1:5
based on the comparison of molar response ratios of these nucleosides
relative to those residues in a hydrolysate of E. coli tRNA
.
(
)Furthermore, the
molar ratio of pseudouridine relative to N
-methylguanosine was determined to be 1:1 based
on comparison of molar response ratios from UV detection compared to
that of authentic standards.
(
)Taken in
toto, the mass spectrometric detection of dihydrouridine and the
molar ratio of one pseudouridine residue per five uridine residues
exactly accounts for the seven uridines predicted from the gene
sequence(33) .
Determination of Base Composition by Electrospray
Mass Spectrometry of RNase T
The RNA 51-mer (8.0 nmol) was
digested with RNase T Hydrolysis Products from the
Region 2423-2473
, and the resultant oligonucleotide
mixture was separated into 25 fractions using DEAE-anion exchange HPLC
(data not shown)(21) . The oligonucleotide fractions were dried
in a vacuum centrifuge, dissolved in H
O, and
rechromatographed by reversed phase HPLC (data not shown) to facilitate
subsequent analysis by electrospray mass spectrometry. For example, the
electrospray mass spectrum of one of the HPLC fractions from the 7-mer
oligonucleotide pool is presented in Fig. 3. Interpretation of
the mass spectrum reveals that five molecular species (an unexpectedly
large number) are present. The exact base composition (and net
modification when present) of each oligonucleotide was determined
directly from each mass spectrometrically measured molecular
mass(21, 34) . The resulting oligonucleotide molecular
weights and compositions are listed in Table 1. The mass spectrum (Fig. 3) is dominated by multiply charged ions associated with
component M
, with ions corresponding to loss of six through
two protons in terms of the neutral oligonucleotide (i.e. ions
(M - 6H)
through (M -
2H)
), from which the value of M was determined to be
2293.33. Based on the experimentally determined molecular mass of this
oligonucleotide and the absence of methylation (see below), the sole
composition and net modification allowed is CUA
Gp + 2H (M
2293.43 (calculated)), which indicates that the
oligonucleotide measured is 2448-AUAACAGp-2454, as dictated by the gene
sequence, and that the single uridine residue has been
posttranscriptionally modified (by reduction) to 5,6-dihydrouridine.
The compositions of all other molecular species present in the
electrospray mass spectrum are also readily determined by accurate mass
measurement and are consistent with sequence elements of the
2448-2454 residue 7-mer. For example, ions of m/z 399.22, 532.59, and 799.44 (Fig. 3) correspond to the (M
- 4H)
through (M - 2H)
ions of an oligonucleotide of M
1600.87. The
composition of this species (M
) is CUA
>p
+ 2H (M
= 1600.98 (calculated), see Table 1). Interestingly, the ion at m/z 538.60
corresponds to the (M - 3H)
charge state of an
oligonucleotide of M
1618.80, which is 18 mass
units (H
O) greater than that of M
(Table 1) and therefore is assigned as CUA
p
+ 2H (M
= 1619.00 (calculated)),
component M
. Similarly, the peak at m/z 636.25 is assigned as the molecular ion of the dinucleotide (U, A)
> p + 2H (M
= 637.38 (calculated),
M
). The ion at m/z 691.28 (M
)
is assigned as the molecular ion, (M - H)
, of
the dinucleotide AGp (M
= 692.43
(calculated)). This assignment is supported by the mass measurement of
the sodium ion adduct, i.e. the replacement of a proton by a
sodium cation (M - 2H + Na)
, at +22 m/z units (M
= 713.29
(measured)). The base compositions of the minor constituents M
through M
imply that they are fragments of the
principal component M
, although it cannot be determined
from the present evidence whether they are formed by nucleolytic
hydrolysis or by gas-phase dissociation of M
ions.
Total Nucleoside Analysis of Oligonucleotide M
An aliquot of the 7-mer sample (0.15 nmol)
was hydrolyzed to its nucleoside constituents using nuclease P 2293.33
and alkaline phosphatase (26) and analyzed by LC/MS to
verify the base composition (CUA
Gp + 2H =
CDA
Gp) determined by molecular mass measurement. The UV
detection chromatogram resulting from this LC/MS analysis is shown in Fig. 4A. The composition of this oligonucleotide was
measured as C
A
G
based on
comparison of molar response ratios from UV detection compared to
authentic standards (data not shown). The mass spectrum recorded at 3.3
min elution time (similar to Fig. 2B, data not shown)
reveals the presence of dihydrouridine, thus confirming the composition
(CDA
G) independently determined directly by mass
measurement. Fig. 4B shows the mass spectrum of the
18.9-min eluant, in which ions of m/z 247, 136, and
268 are detected; these are interpreted as the MH
ion
of dihydrouridine and the BH
and
MH
ions of adenosine, respectively. All three ions
exhibit the same chromatographic profile, with an elution time
different from those of the constituent nucleosides, indicative (35) of a D,A-containing dinucleotide. This result supports the
nucleotide sequence element ADA in the 7-mer.
; m/z 268,
MH
) and D (m/z 247,
MH
).
Total Nucleoside Analysis of Nucleotide Region
2481-2519
Using the same strategy described above, an
RNA 39-mer residue(2481-2519) was also prepared. The yield of RNA
39-mer was determined by UV absorbance at 260 nm as 8.8 nmol. An
aliquot (1/10 of the sample) was enzymatically hydrolyzed to
nucleosides and analyzed by LC/MS (Fig. 5A). Due to an
electronic baseline zero-point malfunction, the true baseline in Fig. 5A was not digitized. Reconstructed ion
chromatograms based on specific m/z values
characteristic of nucleosides of interest are presented in Fig. 5, B-D. In Fig. 5B, the ion
of m/z 245, at 3.6 min elution time, corresponds to
the MH of pseudouridine (
). The other m/z 245 signals at 5.2 and 7.5 min result from a
minor isotope peak of cytidine and the protonated molecular ion
(MH
) of uridine, respectively. The detection of
in the region 2481-2519 is consistent with the recent
identification of pseudouridine at position 2504 by Bakin and
Ofengand(15) . In Fig. 5C, the ion of m/z 258 at 14.5 min elution time corresponds to the
protonated molecular ion (MH
) of
2`-O-methylcytidine. The detection of Cm within the segment
2481-2519 is consistent with previous literature
reports(2, 13, 14) . The ion of m/z 282 at 24.3 min (Fig. 5D)
corresponds to the MH
ion of a monomethyl adenosine.
The assignment specifically as m
A is based on relative
retention time (which is 0.6 min earlier than m
A (28) ) and characteristic UV absorbance ratio (A
/A
).
Detection of Modified Oligonucleotides by
Electrospray Mass Spectrometry of RNase T
The RNA 39-mer
(8.0 nmol) was digested with RNase T Hydrolysis
Products from the Region 2481-2519
, and the resultant
oligonucleotide mixture was separated into 17 fractions using
DEAE-anion exchange HPLC (data not shown)(21) . As described
above, the oligonucleotide fractions were dried in a vacuum centrifuge,
dissolved in H
O, and rechromatographed by reversed phase
HPLC (data not shown) to facilitate subsequent analysis by electrospray
mass spectrometry. Two oligonucleotides were found to contain
posttranscriptionally modified residues, based on accurate mass
measurement. The mass of an oligonucleotide trimer was measured as M
= 1013.0 (spectrum not shown), which
mandates the base composition and net modification as UAGp +
CH
(M
1012.63 (calculated)), which
indicates that the modification-containing oligonucleotide measured
represents 2503-AUGp-2505 as dictated by the gene sequence in this
region. Similarly, an oligonucleotide 7-mer was found to have the base
composition and net modification C
UAGp + CH
(M
2233.63 (measured), 2233.36 (calculated),
data not shown), which indicates that a methylated nucleotide occurs
within the gene sequence-specified fragment 2496-CACCUCGp-2502.
Total Nucleoside Analysis of Oligonucleotide M
An aliquot of the trimer sample (0.16 nmol)
was hydrolyzed to its nucleoside constituents using nuclease
P 1013.0
, venom phosphodiesterase, and alkaline phosphatase (26) and analyzed by LC/MS to verify the base composition (UAGp
+ CH
) determined directly from molecular mass
measurement. Three nucleosides were observed:
, G, and
m
A (data not shown). An additional aliquot of the
hydrolyzed trimer (0.16 nmol) was co-injected with authentic
m
A and analyzed by LC/MS to conclusively demonstrate that
the methyladenosine component is m
A and not the slightly
later eluting isomer m
A(28) . The UV-absorbance
chromatogram corresponding to this analysis (Fig. 6) confirms
the composition established by mass measurement and unambiguously
demonstrates that the methylated adenosine residue from this region of E. coli 23 S rRNA is m
A. Together with gene
sequence information, these experiments demonstrate that this highly
modified trinucleotide has the structure
2502-m
A
Gp-2505.
1013.0 derived from residues
2481-2519 of 23 S rRNA, co-injected with authentic
m
A. UV detection was at 254 nm. Nucleoside assignments
shown are supported by the corresponding mass spectra. A chromatogram
(not shown) produced without addition of m
A appears the
same but without the peak marked as
m
A.
Total Nucleoside Analysis of Oligonucleotide M
An aliquot of the 7-mer sample (0.16 nmol)
was hydrolyzed to its nucleoside constituents using nuclease
P 2233.63
, venom phosphodiesterase, and alkaline phosphatase and
analyzed by LC/MS to verify the base composition (C
UAGp
+ CH
) determined directly from molecular mass
measurement. The composition of this oligonucleotide species was
measured as
C
U
Cm
A
G
based on comparison of molar response ratios from HPLC using UV
detection (Fig. 7) compared to authentic standards. To identify
the nucleoside located 3` to Cm, an additional aliquot (0.40 nmol) of
the 7-mer was digested with RNase T
and alkaline
phosphatase, and the products were fractionated by reversed phase HPLC.
Accurate mass analysis using electrospray ionization (data not shown)
showed that the single dinucleotide had a base composition of C
+ CH
(M
562.10 (measured),
562.43 (calculated)). Enzymatic digestion of this dinucleotide using
venom phosphodiesterase and alkaline phosphatase followed by nucleoside
analysis using LC/MS (35) confirmed the composition of the
dinucleotide as CmC (data not shown). Together with gene sequence
information, these results demonstrate that the M
2233.63 oligonucleotide has the structure 2496-CACmCUCGp-2502.
2233.6 derived from residues
2481-2519 of 23 S rRNA. UV detection was at 254 nm. Nucleoside
assignments shown are supported by the corresponding mass
spectra.
hydrolysis of E. coli 23 S rRNA would yield 913
oligonucleotides, ranging in size from monomers up to two 18-mers. It
is experimentally difficult to chromatographically resolve this large
number of oligonucleotides into subfractions for mass analysis.
Further, inspection of the list of oligonucleotides predicted from the
gene sequence shows the occurrence of several sets of sequence isomers
that would not be distinguished by molecular mass. For example, the
oligonucleotide 7-mers 1260-ACAUAAG-1266, 1675-CAAAAUG-1681, and
1744-AAAUCAG-1750 are isomers of 2448-AUAACAG-2454 (from the peptidyl
transferase region). To eliminate compositional redundancy and
facilitate the preparation of oligonucleotides for analysis by mass
spectrometry, we have used an approach based on nuclease protection of
a heteroduplex formed between a selected region of rRNA and a
complementary oligodeoxyribonucleotide, followed by enzymatic
degradation of the DNA and isolation of the remaining RNA fragment.
G-2445 and
-2257) that serve as markers. Similarly,
an RNA 39-mer (residues 2481-2519) was chosen for analysis
because of the density of modifications as well as the putative
existence (based on reverse transcriptase data) of an unknown
modification at position 2501(15) .
G, D,
(two sites), Cm, and m
A. The
sequence locations of m
G and
in the 2423-2473
segment were not sought because their presence is consistent with
earlier work that placed them at 2445 (14, 15) and
2457(15) , respectively. The remaining modifications were
structurally characterized at the nucleoside level and placed
specifically at sites previously determined to be modified:
D-2449(39) , Cm-2498(2, 13, 14) ,
m
A-2503(14) , and
-2504(15) .
had revealed an unusual oligonucleotide, (A,U)AACAGp,
which released a modified dinucleotide AUp upon digestion with RNase
A(39, 40) . The electrophoretic mobility of this
modified dinucleotide differed from that of A
p, implying that the
modified residue is not
; subsequent sequencing studies of RNase
T
hydrolysis products indicated modification of U-2449 and
not A-2450(12) . Furthermore, no methyl-containing
oligonucleotides were known to occur at this position on the
fingerprint(11) . Inspection of the E. coli 23 S rDNA
sequence (33) reveals that only a single oligonucleotide having
the sequence AUAACAG occurs, at position 2448-2454, which is
shown by secondary structural models to be located in the universally
conserved, single-stranded central loop of domain
V(2, 13) . The determination of Cm-2498 and
-2504 agrees with recent assignments(14, 15) ,
but the characterization of m
A-2503 differs from the
previous report of its identity as m
A(14) . The
latter incorrect assignment is evidently due to the closeness of HPLC
retention times (30) required to distinguish the two isomers.
hydrolysis product 2496-CACmCUCGp-2502, followed by accurate
molecular mass measurement and nucleoside analysis by LC/MS, clearly
shows the lack of modification at position 2501, a result that has been
independently repeated.
(
)Substoichiometric
occurrence of modification at this position cannot be rigorously
excluded nor can degradation of modification as a consequence of
chemical instability. However, there were no unaccounted for
modifications evident in digests of total 23 S RNA (Fig. 1) or
of the domain V 39-mer 2481-2519 (Fig. 5).
(37) and
tRNA
(43) . NMR studies of the nucleoside (44) as well as crystallographic data from the nucleoside (44, 45) and tRNA (46) indicate a
conformational preference in the sugar for the inherently more flexible
C2`-endo rather than the more common C3`-endo form
found in the base-stacked, ordered conformation of the A-type RNA
helix. Compared with C3`-endo, the C2`-endo conformation is inherently more flexible (47) with a wider
range of allowable glycosyl torsion angles(48) , a property
which would influence nucleotides adjacent to D-2449. The question of
whether dihydrouridine occurs in other 23 S RNAs and is
phylogenetically conserved at position 2449 is presently unknown and is
under investigation.
G-2069(13) ,
m
G-2445(14, 15) , D-2449 (see text),
-2457(15) ,
Cm-2498(2, 13, 14) , m
A-2503 (see
text),
-2504, Um-2552,
-2580, and
-2605(15) .
A, 2-methyladenosine;
m
A, N
-methyladenosine; Cm,
2`-O-methylcytidine; LC/MS, combined high performance liquid
chromatography-mass spectrometry; M, neutral molecule;
MH
, protonated molecule;
BH
, fragment ion corresponding to the
protonated free base of a nucleoside.
We thank P. F. Crain, R. F. Gesteland, and S. C.
Pomerantz for helpful discussions, J. M. Peltier for providing HPLC
molar response data, and J. L. Stauffer for engineering expertise.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.