©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Posttranscriptional Modification of the Central Loop of Domain V in Escherichia coli 23 S Ribosomal RNA (*)

(Received for publication, March 14, 1995)

Jeffrey A. Kowalak (1)(§) Eveline Bruenger (2) James A. McCloskey (1) (2)(¶)

From the  (1)Departments of Biochemistry and (2)Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84132

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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-mAGp-2505.


INTRODUCTION

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) .


MATERIALS AND METHODS

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 NHOAc, 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% CHCN to 1.0 M triethylammonium bicarbonate, pH 7.8, 20% CHCN 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% CHCN and CHCN:HO (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 HO and diluted with CHOH 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) .


Figure 3: Electrospray mass spectrum of oligoribonucleotide 7-mer produced from RNase T 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.






RESULTS

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 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.


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 (); 3, unknown (U*); 4, 5-methylcytidine; 5, 2`-O-methylcytidine (Cm); 6, 5-methyluridine (mU); 7, 7-methylguanosine (mG); 8, 2`-O-methyluridine (Um); 9, adenosine 2`,3`-cyclic phosphate (A>p); 10, 1-methylguanosine; 11, 2`-O-methylguanosine; 12, N-methylguanosine (mG); 13, dinucleotide of component 3 and A; 14, 2-methyladenosine (mA); 15, N-methyladenosine (mA). 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.



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 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 (mU) 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.

The detection of 2-methyladenosine and N-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 (mA) and 25.3 min (mA) 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 mA and mA are present in E. coli 23 S rRNA. Determination of the sequence location of mA 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).


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 (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 (mG). Peaks denoted by triangles are from background ions.



Inspection of the E. coli 23 S rDNA sequence shows that the unmodified base composition of the 51-mer is CUAG. 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 Hydrolysis Products from the Region 2423-2473

The RNA 51-mer (8.0 nmol) was digested with RNase T, 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 HO, 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 CUAGp + 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 (HO) greater than that of M (Table 1) and therefore is assigned as CUAp + 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 2293.33

An aliquot of the 7-mer sample (0.15 nmol) was hydrolyzed to its nucleoside constituents using nuclease P and alkaline phosphatase (26) and analyzed by LC/MS to verify the base composition (CUAGp + 2H = CDAGp) 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 CAG 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 (CDAG) 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.


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; 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 mA is based on relative retention time (which is 0.6 min earlier than mA (28) ) and characteristic UV absorbance ratio (A/A).


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.



Detection of Modified Oligonucleotides by Electrospray Mass Spectrometry of RNase T Hydrolysis Products from the Region 2481-2519

The RNA 39-mer (8.0 nmol) was digested with RNase T, 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 HO, 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 CUAGp + 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 1013.0

An aliquot of the trimer sample (0.16 nmol) was hydrolyzed to its nucleoside constituents using nuclease P, 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 mA (data not shown). An additional aliquot of the hydrolyzed trimer (0.16 nmol) was co-injected with authentic mA and analyzed by LC/MS to conclusively demonstrate that the methyladenosine component is mA and not the slightly later eluting isomer mA(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 mA. Together with gene sequence information, these experiments demonstrate that this highly modified trinucleotide has the structure 2502-mAGp-2505.


Figure 6: HPLC analysis of an enzymatic digest of oligonucleotide M 1013.0 derived from residues 2481-2519 of 23 S rRNA, co-injected with authentic mA. UV detection was at 254 nm. Nucleoside assignments shown are supported by the corresponding mass spectra. A chromatogram (not shown) produced without addition of mA appears the same but without the peak marked as mA.



Total Nucleoside Analysis of Oligonucleotide M 2233.63

An aliquot of the 7-mer sample (0.16 nmol) was hydrolyzed to its nucleoside constituents using nuclease P, venom phosphodiesterase, and alkaline phosphatase and analyzed by LC/MS to verify the base composition (CUAGp + CH) determined directly from molecular mass measurement. The composition of this oligonucleotide species was measured as CUCmAG 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.


Figure 7: HPLC analysis of an enzymatic digest of oligonucleotide M 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.




DISCUSSION

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 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.

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 (mG-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) .

In the present study, six sites of posttranscriptional modification were determined by mass spectrometry in the two segments of domain V that were studied: mG, D, (two sites), Cm, and mA. The sequence locations of mG 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) , mA-2503(14) , and -2504(15) .

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 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 Ap, 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 mA-2503 differs from the previous report of its identity as mA(14) . The latter incorrect assignment is evidently due to the closeness of HPLC retention times (30) required to distinguish the two isomers.

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 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).

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(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.


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: mG-2069(13) , mG-2445(14, 15) , D-2449 (see text), -2457(15) , Cm-2498(2, 13, 14) , mA-2503 (see text), -2504, Um-2552, -2580, and -2605(15) .




FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM29812. The University of Utah Protein/DNA Core Facility is supported by Grant 5P30CA42014 from NCI, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Biochemistry, SJ-70, University of Washington, Seattle, WA 98195.

To whom correspondence and reprint requests should be addressed: 311A Skaggs Hall, University of Utah, Salt Lake City, UT 84112. Tel.: 801-581-5581; Fax: 801-581-7457.

The abbreviations used are: HPLC, high performance liquid chromatography; mA, 2-methyladenosine; mA, 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.

J. A. McCloskey, unpublished results.

J. M. Peltier, unpublished results.

E. Bruenger and P. F. Crain, unpublished experiments.


ACKNOWLEDGEMENTS

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.


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