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Originally published In Press as doi:10.1074/jbc.M600377200 on May 26, 2006

J. Biol. Chem., Vol. 281, Issue 31, 22108-22117, August 4, 2006
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Modifications in Thermus thermophilus 23 S Ribosomal RNA Are Centered in Regions of RNA-RNA Contact*

Jonas Mengel-Jørgensen1, Søren Skov Jensen, Anette Rasmussen, Jacob Poehlsgaard, Jens Jørgen Lønsmann Iversen, and Finn Kirpekar2

From the Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark

Received for publication, January 13, 2006 , and in revised form, May 24, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ribosomal RNA from all organisms contains post-transcriptionally modified nucleotides whose function is far from clear. To gain insight into the molecular interactions of modified nucleotides, we investigated the modification status of Thermus thermophilus 5 S and 23 S ribosomal RNA by mass spectrometry and chemical derivatization/primer extension. A total of eleven modified nucleotides was found in 23 S rRNA, of which eight were singly methylated nucleotides and three were pseudouridines. These modified nucleotides were mapped into the published three-dimensional ribosome structure. Seven of the modified nucleotides located to domain IV, and four modified nucleotides located to domain V of the 23 S rRNA. All posttranscriptionally modified nucleotides map in the center of the ribosome, and none of them are in contact with ribosomal proteins. All except one of the modified nucleotides were found in secondary structure elements of the 23 S ribosomal RNA that contact either 16 S ribosomal RNA or transfer RNA, with five of these nucleotides physically involved in intermolecular RNA-RNA bridges. These findings strongly suggest that the post-transcriptional modifications play a role in modulating intermolecular RNA-RNA contacts, which is the first suggestion on a specific function of endogenous ribosomal RNA modifications.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
All cellular protein synthesis is performed by ribosomes, which are large ribonucleoprotein particles. The prokaryote ribosome consists of two stable and separable entities, a 50 S and a 30 S subunit. The 50 S subunit contains two rRNAs of ~3000 and 120 nucleotides (23 S and 5 S rRNA, respectively) and around 35 proteins, whereas the 30 S subunit contains 16 S rRNA of ~1600 nucleotides and around 20 proteins; the exact numbers vary with the species. Eukaryotic ribosomes are larger, but structural features are remarkably conserved between the domains of life.

rRNAs are post-transcriptionally modified at specific nucleotides, but the number of modified nucleotides varies greatly. The large ribosomal RNA in mitochondria contains just a few modified nucleotides (1), Escherichia coli 23 S rRNA has 25 (2, 3), whereas over 100 are found in vertebrate cytoplasmic 28 S rRNA (4). The function of post-transcriptional rRNA modifications is far from clear, although they have been implicated in various processes. Specific rRNA methylation is used by numerous antibiotics-producing microorganisms as a means of autoprotection (see e.g. Refs. 5 and 6), but only a small fraction of post-transcriptional modifications can be assigned to this well defined function. E. coli 23 S rRNA modifications cluster in functionally principal parts of the ribosome such as the peptidyl transferase center and inter-subunit bridges when modeled into the structure of the Haloarcula marismortui large ribosomal subunit (7). The modified nucleotides in the central domains of 23 S rRNA from H. marismortui itself are all located in regions of intra- or intermolecular RNA-RNA contact (8) suggesting structure stabilization.

The post-transcriptionally modified rRNAs are essential for assembly of functional E. coli ribosomal subunits (9, 10). This is in contrast to Bacillus stearothermophilus, where functional ribosomes can be assembled with in vitro transcribed 23 S rRNA, albeit less efficiently than with authentic 23 S rRNA (11). Inactivation of the modifying enzymes in E. coli has been reported to impair protein synthesis and cell growth (12-14), but recent data suggest that these effects may in some cases be ascribed to (unknown) secondary functions of these enzymes, not to the rRNA modification itself (15, 16). A ribose methylation of G2270 (E. coli numbering 2251) is required for assembly of the large and small ribosomal subunits in yeast mitochondria (1), and various 25 S rRNA pseudouridines ({Psi})3 are required for optimal translation in yeast (17). On the other hand, systematic elimination of single ribose methylations in yeast rRNA does not affect cell growth (18). In summary, there is no coherent picture of the significance and function of post-transcriptional rRNA modifications.

Because it is now well established that the 23 S rRNA constitutes the peptidyl transferase activity with numerous modified nucleotides but no proteins located in the active site (19, 20), the spatial localization and molecular interactions of modified rRNA nucleotides become increasingly important to analyze. The availability of moderate to high resolution x-ray crystallography structures of ribosomes and ribosomal subunits from various prokaryotes (19-23) yields a unique opportunity to view post-transcriptional rRNA modifications in their three-dimensional context and, thereby, gain insight on their function. We searched for modified nucleotides in the 23 S rRNA from Thermus thermophilus, since the three-dimensional structure of the entire ribosome was first published for this organism (19). Hence, all modified rRNA nucleotides can be viewed in their three-dimensional surroundings. Most of the modifications were found directly by mass spectrometry (MS), but the mass-silent modification pseudouridine was located by chemical modification with a carbodiimide reagent followed by reverse transcriptase primer extension (24). A total of 11 modified nucleotides were found and were fitted into the three-dimensional structure of the T. thermophilus ribosome (19). The function of the modified nucleotides is discussed in light of their placement.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of rRNA—T. thermophilus (strain HB8) was grown as batch culture in a 3-liter BTS-05 bioreactor (Applicon, B.V., Schiedam, the Netherlands) at 72 °C and pH-controlled at 7.5. The growth medium contained 4 g/liter yeast extract, 8 g/liter polypeptone, and 2 g/liter NaCl with maize oil as antifoam. Ribosomal subunits from 30-g dry weight of harvested cells were prepared via sucrose gradients as described (25). The fractions containing the large ribosomal subunit were pooled, and the RNA-protein complex was precipitated with 2.5 volumes of ethanol. The RNA was purified by three rounds of extraction with a 1:1-mixture of phenol/chloroform and recovered by ethanol precipitation. 5 S rRNA was separated from 23 S rRNA by ultrafiltration through a Microcon YM-100 membrane filter (Millipore) as described by the manufacturer. The flow-through contained the 5 S rRNA. 23 S rRNA was isolated from 5 S rRNA by gel filtration through a Microspin S400 HR column (Amersham Biosciences) as recommended by the manufacturer.

Details on the site-directed mung bean nuclease protection for isolation of rRNA subfragments can be found in a previous study (2). Briefly, oligodeoxynucleotides complementary to T. thermophilus 23 S rRNA position 2505-2568 (E. coli 2492-2557) or 2240-2289 (E. coli 2229-2278) were annealed to the 23 S rRNA followed by digestion with a combination of mung bean nuclease and RNase A to degrade single-stranded nucleic acids. The digestion mixture was separated on a denaturing polyacrylamide gel and stained with ethidium bromide. The band corresponding to the protected 23 S rRNA subfragment was cut out and eluted.

RNase Digestion— 6 pmol of 5 S rRNA was digested with either 0.4 µg of RNase A (Sigma) or 10 units of RNase T1 (U. S. Biochemical Corp.), and 2 pmol of 23 S rRNA was digested with either 3 µg of RNase A or 100 units of RNase T1 as described (26). 0.5 volume of 0.5 M HCl was added to the RNase T1 digestions to hydrolyze 2',3'-cyclic phosphates with incubation at room temperature for 30 min. The HCl was removed under vacuum after addition of 4 volumes of H2O.

The 23 S rRNA digestions were size-separated by in-house fabricated micro reverse-phase columns (5) with minor adjustments. Poros 50R3 (Perseptive Biosystems) was used as column material, and columns were equilibrated and washed with 10 mM triethylammonium acetate (TEAA) pH 7.0. RNase A digestions were eluted in two steps using 8% acetonitrile (MeCN)/10 mM TEAA and 15% MeCN/10 mM TEAA, respectively. The first elution buffer contained 6% MeCN/10 mM TEAA for the RNase T1 digestions. The eluates were dried and dissolved in water to a final concentration of to 2 pmol of intact rRNA per microliter.

Mass Spectrometry and Data Analysis—MALDI mass spectrometry was performed as previously described (26) using 3-hydroxypicolinic acid as matrix on either a Bruker Reflex IV (Bruker-Daltonics, Bremen, Germany) or a Perseptive Voyager STR (PerSeptive Biosystems, Framingham, MA) instrument in positive ion reflector mode, using delayed ion extraction. The spectra were processed using the "m/z" free software (Proteomics Ltd., New York, NY) with three-point internal calibration on predicted unmodified digestion fragments.

Theoretical digestions of rRNA were done using a "Mongo Oligo Mass Calculator" (www-medstat.med.utah.edu/massspec/mongo.htm). The calculated compositions from observed masses were obtained by using an "Oligo Composition Calculator" (medstat.med.utah.edu/masspec/compo.htm) with the following parameters: a mass accuracy of ±0.005%, one to four methyl groups, and one to three guanosine nucleotides for RNase T1 digestions or one to three pyrimidine nucleotides for RNase A digestions. Matching of nucleotide compositions to the T. thermophilus 23 S rRNA sequence was done with the GPMAW software (Lighthouse Data, Odense, Denmark).

MALDI quadrupole time-of-flight (MicroMass, Manchester, UK) tandem mass spectrometry on positive ions was performed as reported (27). Nanoelectrospray ionization on a Bruker Esquire electrospray ion trap instrument (Bruker-Daltonics, Bremen, Germany) was performed in negative ion mode. These samples were dissolved in 50% MeCN/10 mM TEAA to a concentration of 1-2 pmol/µl. The nanoelectrospray needles (Proxeon, Odense, Denmark) were washed with 80% formic acid prior to sample loading to reduce cation salt formation.

Sequencing of 23 S/5 S rRNA Operons—T. thermophilus has two operons coding for 23 S and 5 S rRNA. The two operons have identical 5'-promoter regions but different 3'-downstream regions (28). Two PCRs were performed with the common promoter-specific primer 5'-AGCGGGGGATCTTGAAAGCTGGGGT-3' (-54 to -30 upstream of the coding region) and the two different downstream primers 5'-TGTCTAGAAGCTTCACCTTATCCCCCTTTGC-3' (complementary to position 3270-3300 in the region termed "675" in Ref. 28) or 5'-ACGCGCCCACCTGGAGGTGT-3' (complementary to position 3273-3292 in the region termed "700" in Ref. 28) to amplify the two operons. The DNA template was obtained from a T. thermophilus colony that was suspended in water and boiled. The desired PCR products were purified via agarose gels and sequenced using a "Big Dye" terminator cycle sequencing kit (Applied Biosystems). Regions of 23 S rDNA that did not correspond to the sequence published by Hopfl et al. (29) (GenBankTM accession number X12612 [GenBank] ) were sequenced on both strands. GenBankTM accession numbers for the two T. thermophilus 23 S/5S operons are AY379548 [GenBank] and AY379549 [GenBank] , respectively.


Figure 1
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  		FIGURE 1.
RNase digestion of T. thermophilus 23 S rRNA analyzed by MALDI TOF mass spectrometry. A, RNase A digestion, low mass window. B, RNase T1 digestion, high mass window. Boxed m/z values represent digestion fragments not expected from gene sequence-based theoretical digestion.

 
Structure Modulation of T. thermophilus Ribosome—The coordinates for the structure of the T. thermophilus ribosome reported in a previous study (19) can be found in the Protein Data Bank (accession 1GIY [PDB] ). The MOLMOL software (30) was used to investigate the ribosome structure around the methylated nucleotides and to graphically highlight these nucleotides.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Sequencing of T. thermophilus 23 S/5 S rDNA Genes—Our strategy for mapping post-transcriptional modifications is based on complete digestion of the RNA with a nucleotide-specific RNase and subsequent analysis of the digestion mixture by MALDI-TOF mass spectrometry. Fragments harboring post-transcriptional modifications are identified by deviations between the observed fragment masses and the masses calculated from the gene sequence (26). We therefore needed the exact sequence of the T. thermophilus large ribosomal subunit RNA genes. T. thermophilus has two operons each encompassing genes for 23 S rRNA, 5 S rRNA, and tRNAGly. After PCR amplification, we sequenced each of the 23 S/5 S rDNA operons and found them identical. Compared with the previously published 23 S rDNA (29) (GenBankTM accession number X12612 [GenBank] ), G445 and G1180 were absent and an extra Gly at position 519 was present in our version of T. thermophilus strain HB8. The 5 S rDNA was identical to the previously published sequences (28).

Detection of 23 S rRNA Post-transcriptional Modifications by Mass Spectrometry—rRNA from the large ribosomal subunit was purified via isolation of the ribosomes followed by separation of the ribosomal subunits by centrifugation through sucrose density gradients. The 23 S/5 S rRNA was recovered by phenol/chloroform extraction, and the two rRNA species were size-separated by gel filtration or ultrafiltration.

The 5 S rRNA was digested to completion with either RNase A or RNase T1, and the digestions were mass analyzed (data not shown). We did not detect any post-transcriptional modifications in 5 S rRNA, which is in accordance with absence of post-transcriptional modifications in all other bacterial 5 S rRNA investigated. The only unexpected observation was the presence of a mixed population of 5 S rRNAs with either a single or two A-nucleotides at the 5'-end.

T. thermophilus 23 S rRNA was likewise digested to completion with either RNase A or RNase T1. The digestions were additionally separated into two fractions by reverse phase chromatography. This was necessary mainly to reduce the amount of mono- and dinucleotides that otherwise tend to form gas-phase clusters appearing in the m/z regions of less abundant digestion fragments of larger size. Fig. 1A shows the low mass region of the RNase A digestion. Four fragments, at m/z 990.14, 1671.30, 1718.24, and 1992.33 did not correspond to calculated digestion fragments, and are therefore likely to harbor a post-transcriptional modification. No further potentially modified fragments were identified in the higher m/z range after RNase A digestion (data not shown). Fig. 1B shows the high mass region of the RNase T1 digestion; here a single candidate for a post-transcriptionally modified fragment appeared at m/z 3565.51. Spectra from the lower m/z regions indicated candidates for post-transcriptionally modified fragments at m/z 723.16, 1013.16, 1623.21, and 3213.32 after RNase T1 cleavage (Table 1).


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  		TABLE 1
Modified nucleotides in T. thermophilus 23 S rRNA as identified by RNase digestion, MS, and MS/MS

 


Figure 2
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  		FIGURE 2.
Electrospray ionization ion trap tandem MS spectra of post-transcriptionally modified RNase digestion fragments. A, MS/MS spectrum of doubly deprotonated RNase T1 digestion fragment with the calculated composition A, C2, G, U, and Me. Sequence interpreted from the spectrum is given; see text for details. B, MS3 spectrum derived from the c2-ion of the singly deprotonated RNase A digestion fragment with calculated composition G, U2, and Me. Interpretation of the spectrum is given; see text for details.

 
Determination of Modified Nucleotides—McCloskey and co-workers have reported how the nucleotide composition and methylation level of RNase T1 digestion products can be predicted as a function of mass accuracy (31). Methylation is by far the most common mass-increasing modification in rRNA. We therefore calculated the possible nucleotide composition of all the above fragments potentially harboring post-transcriptionally modified nucleotides assuming a mass accuracy of ±0.005%, allowing one to four methyl groups and up to two sites where cleavage after the normal target nucleotides is prevented due to methylation. The calculations gave unique composition for all digestion fragments, but for the two longest RNase T1 fragments, which yielded three and four possible compositions, respectively. We then compared potential compositions of all the modified digestion fragments to the T. thermophilus 23 S rRNA sequence, also taking the enzyme specificities into account. An RNase substrate nucleotide was assumed in the 3'-end of the fragment, and methylations were assumed on internal, uncleaved substrate nucleotides. Only the largest modified RNase T1 fragment (m/z 3565.51) could be located on the T. thermophilus 23 S by this strategy, here to position 1932-1942. All the remaining smaller modified RNase fragments could have originated from numerous positions in the 23 S rRNA, and an exact location could therefore not be assigned.


Figure 3
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  		FIGURE 3.
MALDI quadrupole TOF MS/MS spectrum of RNase T1 digestion fragment at m/z 3213.44. Sequence interpretation is indicated; c-type fragments marked with an asterisk are derived from y8-ions as explained in the text.

 
We subsequently performed tandem mass spectrometry on the post-transcriptionally modified (i.e. methylated) fragments to obtain a sequence and information on the exact location of the modification; the predicted compositions were taken into account in tandem mass spectrum interpretations. Fig. 2A shows an electrospray ionization-ion trap MS/MS mass spectrum of the doubly deprotonated RNase T1 digestion fragment with the predicted composition (A, C2, G, U, and Me). The sequence 5'-AC[C+Me]UG-3' can be read from the c- and y-ion series (nomenclature according to Ref. 32), and the methyl group must be on the nucleobase, because a distinct signal corresponding to neutral loss of a methylated cytosine is present at m/z 748.0. An RNase T1 digestion fragment with the deduced sequence 5'-ACCUG-3' preceded by a G-nucleotide is present only once in T. thermophilus 23 S rRNA, and we can therefore assign the methylated C to position 1983. An example of MS3 to disclose a methyl group on the ribose is shown in Fig. 2B. The MS/MS results of the RNase A digestion fragment (G, U2, and Me) revealed that the methyl group was present on a tandem MS fragment that harbored the first two nucleotides (a c2-fragment; not shown). Using this fragment as precursor for MS3 resulted in signals corresponding to an unmodified G-nucleotide (x1) and the loss of an unmodified uracil-base (M-UH)-, leaving the ribose of the uridine-nucleotide as the only possible site for methylation.

We obtained better tandem MS results on the larger digestion products with a MALDI-quadrupole-TOF instrument, exemplified in Fig. 3 with the m/z 3213.38 species from the RNase T1 digestion as precursor ion. A series of backbone fragment ions (dominated by y-ions) plus a series of y8-ions that have lost consecutive nucleotides from the 3'-end allowed complete reading of the sequence. The two methyl groups were assigned to U1960 and C1963, respectively. By a similar tandem MS approach, we assigned the two methyl groups in the m/z 3565.51 RNase T1 digestion fragment to U1937 and C1941. The C1941-methylation is on the ribose, deduced from tandem MS of the unique overlapping RNase A fragment at m/z 1992.33. The sequence of the modified digestion fragments together with the sites of methylations as determined by tandem MS are summarized in Table 1. Assuming that the RNase T1 fragment [Gm]G overlaps with the RNase A fragment G[Gm]GGC, and that the RNase A fragment GAA[AU+Me] overlaps with the RNase T1 fragment AAA[U+Me]UC[C+Me]UUG, we found eight nucleotide monomethylations in the 23 S rRNA from T. thermophilus.

Three of the methylated nucleotides could not be assigned to a specific position in the 23 S rRNA, because their sequence contexts were present in numerous copies. These were [Um]GU, [mA]UG, and G[Gm]GGC. However, comparison with modification patterns in other organisms gives suggestions to the location of these modifications. E. coli and a number of other species harbor ribose-methylated uridine at position 2552 (E. coli numbering) in the sequence context [Um]GU, identical to the above (1, 3, 4, 7, 33). Likewise, A2503 (E. coli numbering) in the sequence AUG is methylated in E. coli (3) and in Bacillus megaterium.4 The two equivalent positions in T. thermophilus are U2563 and A2514, respectively. We therefore isolated the T. thermophilus position 2505-2568 (E. coli. 2494-2557) 23 S rRNA subfragment by site-directed mung bean nuclease protection (2). Parallel RNase A and RNase T1 digestions followed by mass spectrometry revealed the expected [mA]UG and [Um]GU fragments (data not shown), demonstrating that the T. thermophilus 23 S rRNA harbors a basemethylated A2514 (E. coli 2503) and a ribose-methylated U2563 (E. coli 2552). Using the above logic (1, 3, 33) and approach, the ribose methylation in G[Gm]GGC was located to position 2263 (E. coli 2252). The modifications and means of identification are summarized in Table 1.

Detection of Pseudouridines—Pseudouridine is a structural isomer of uridine and is therefore a mass-neutral post-transcriptional modification. We screened T. thermophilus 23 S rRNA for {Psi}s by the CMCT modification method. CMCT reacts with the N3 position in uracil, the N1 position in guanine, and both the N1 and N3 positions in {Psi}. Subsequent mild alkaline treatment of the RNA strips off the CMCT from all positions but N3 in {Psi}, which yields a distinct stop at the 3'-adjacent nucleotide by primer extension analysis of the RNA (24). More than 98% of the T. thermophilus 23 S rRNA was screened for {Psi}s by this method. Unambiguous CMCT-dependent primer extension stops were observed at positions U1932 (E. coli 1911), U1938 (E. coli 1917) (Fig. 4), and U2616 (E. coli 2605); all three positions are also {Psi}sin E. coli. Fig. 4 also reveals a strong primer extension stop at position 1936 (E. coli 1915), where T. thermophilus contains a methylated uridine and E. coli harbors an m3{Psi}. It was, however, not possible to discern whether the stop was CMCT-dependent or not.


Figure 4
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  		FIGURE 4.
Autoradiograms showing CMCT/alkali-treatment followed by primer extension analysis to detect pseudouridines. C, U, A, and G indicate RNA sequencing lanes. Samples in the first and third lanes from the right were treated with alkali for 4 h, whereas those in the second and fourth lanes from the right were similarly treated for 2 h. Modified nucleotides are indicated by their position in T. thermophilus 23 S rRNA.

 
Less intense CMCT-dependent primer extension stops were observed at positions 918, 2201, and 2210 (E. coli 871, 2180, and 2189, respectively). It was recently shown in studies on H. marismortui 23 S rRNA that weak CMCT-dependent stops are often false positives with regard to {Psi} (8, 34). We therefore investigated these putative {Psi}s using an independent method. rRNA subfragments were isolated by site-directed mung bean nuclease protection, and these subfragments were cyanoethylated by treatment with acrylonitrile, which specifically modifies N1 on {Psi}s, resulting in a 53.0-Da mass increment (27). The subfragments were digested with RNase A and/or RNase T1, and the digests were subjected to MALDI mass spectrometry, but none of the positions 918, 2201, or 2210 (E. coli 871, 2180, and 2189, respectively) could be verified as {Psi}s, because none of the relevant digestion products exhibited the 53.0-Da mass shift (data not shown). These nucleotides are therefore considered unmodified. The data on {Psi}s are summarized in Table 2.


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  		TABLE 2
{Psi}s in T. thermophilus 23 S rRNA as identified by CMCT modification and primer extension

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
A combination of mass spectrometric and biochemical approaches identified 11 post-transcriptionally modified nucleotides in 23 S from T. thermophilus (see Tables 1 and 2). Eight modifications were single methylations, and three were {Psi}s.

A relevant issue is the exhaustiveness of our detection/identification of post-transcriptional modifications. The search for {Psi}s interrogated nearly the entire 23 S rRNA and can essentially be considered complete. Three {Psi}s being found is comparable to the number in H. marismortui and Deinococcus radiodurans (34) but significantly less than the eight {Psi}s present in E. coli (3). The MALDI-TOF technique makes detection of a single modified digestion fragment hard if it is located among a large molar excess of other ions. In particular, post-transcriptional modifications that remain in mono- or dinucleotides both after RNase A and RNase T1 digestions are difficult to identify ab initio, because this m/z range is dominated by matrix signals, a large excess of unmodified digestion products, and various adducts of both. 184 nucleotides or around 6% of the 23 S rRNA nucleotides remain in mono- or dinucleotides regardless of the RNase used. Furthermore, modified RNase fragments that coincide in mass with an unmodified fragment or various adducts of these will remain undetected, although the former scenario is rare as demonstrated by earlier calculations (31). We do not have an estimate on the number of missed modifications, but there are most likely more post-transcriptional modifications in T. thermophilus 23 S rRNA to be identified.

Seven of the modified nucleotides mapped to domain IV of the 23 S rRNA secondary structure, and four were found in domain V (Fig. 5A). No modifications were detected in domain II of T. thermophilus 23 S rRNA, which is noteworthy because most other organisms harbor post-transcriptional modifications in this domain. Our data strongly suggest that the hypermodified helix 35 of E. coli containing m1G745, {Psi}746, and m5U747 has no modified counterparts in T. thermophilus. This is surprising because an m1G is found at either position 745 or 748 (E. coli numbering) in a very broad range of bacteria (35). Table 3 gives an overview of which other organisms (potentially) have modified nucleotides in secondary-structure positions equivalent to those found in T. thermophilus; the table is not complete, but phylogenetically representative in view of the existing literature. It should also be noted that many of the modifications in eukaryotes and archaea are putative, because only the small nucleolar RNAs specific for given modifications have been identified, whereas the modifications themselves have not been experimentally confirmed. Some of the modification positions listed in Table 3 are conserved in a wide variety of organisms. Most notable are methyl({Psi})1936, {Psi}1938, and Um2563 (E. coli 1915, 1917, and 2552), represented by organisms from all three domains of life: eukaryotes, eubacteria, and archaea. {Psi}1932 and Gm2262 (E. coli 1911 and 2251) are restricted to eubacteria/organelles and eukaryotes, whereas mA2514 and {Psi}2616 (E. coli 2503 and 2605) modifications appear specifically absent in eukaryotes. The remaining four modified nucleotides, methylated C1941, U1960, C1963, and C1983 (E. coli 1920, 1939, 1942, and 1962) are only found in a single or very few other organisms. Given that the function of ribosomal RNA modifications is largely unknown, and that some appear dispensable (15, 16), it is intriguing that small nucleolar RNAs directing ribose methylation were identified in Pyrococcus species (36, 37) and Arabidopsis thaliana (38) at positions equivalent to T. thermophilus C1941 (E. coli 1920) and C1983 (E. coli 1962), respectively. Because the two former organisms are completely unrelated to T. thermophilus and synthesize rRNA modifications by a very different mechanism, it is likely that these modifications are specifically important in the named organisms. Targeted gene disruption experiments may shed light on the function of these post-transcriptional modifications.


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  		TABLE 3
T. thermophilus 23 S rRNA modification positions found in other organisms Data were compiled from other studies (1, 3, 4, 7, 8, 18, 33, 34, 36-38, 50-58).

 


Figure 5
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  		FIGURE 5.
Location of identified nucleotide modifications. A, mapping in the secondary structure of the T. thermophilus 23 S rRNA. The outline is the secondary structure of the 3'-half of T. thermophilus 23 S rRNA, with enlarged views of the regions where the modified nucleotides are located. B, mapping in the three-dimensional structure of T. thermophilus ribosome. The large picture is a view from the ribosomes right-hand side of a slice with a thickness of less than the combined sizes of tRNAs in both the A- and P-sites. The inset shows an interface view of the 50 S subunit indicating the thickness of the abovementioned ribosomal slice. Gray strings: backbone of large ribosomal subunit rRNAs. Gray outline: contour of the large ribosomal subunit. Golden strings: backbone of 16 S rRNA. Golden outline: contour of the small ribosomal subunit. Blue ribbon: tRNA in the P-site. Magenta ribbon: tRNA in the A-site. Red spheres: pseudouridines in 23 S rRNA. Yellow spheres: methylated nucleotides in 23 S rRNA. The nucleotides are given with E. coli numbering.

 
Several reports indicate that certain nucleotide modifications play a role in thermal stabilization of RNA structure. The number of ribose-methylated nucleotides increases with increasing growth temperature for Sulfolobus solfataricus rRNA (39) and Bacillus stearothermophilus (40), and hyperthermophilic Methanococci have a higher content of tRNA ribose-methylated nucleotides than mesophilic ditto (41). Ribose methylation (42), 5-methylation of U (43) and C (44), as well as {Psi}s (45) raise the thermal stability of RNA structures. Although the present study generally does not position the post-transcriptional modifications at the atomic level, the six completely characterized modifications are of the thermo-stabilizing nature: three ribose methylations and three {Psi}s. The base methylated U1960 and C1983 are tentatively also thermo-stabilizing methylations as counterparts to the homologous m5U1939 and m5C1962 in E. coli (Table 1). These types of post-transcriptional modifications are in good agreement with the fact that T. thermophilus has optimal growth at 75 °C.

We mapped the modified nucleotides in the three-dimensional structure of the ribosome (19). The structure is at 5.5-Å resolution, allowing tracing of the phosphodiester backbone of rRNAs (and polypeptide backbone of proteins) but not of individual nucleobases. Careful analysis of the available x-ray coordinates revealed that none of the modified nucleotides appear in contact with ribosomal proteins, suggesting that post-transcriptional rRNA modifications generally do not have a function in intermolecular interactions between rRNA and protein. Fig. 5B shows a slice of the ribosome with a thickness of less than tRNAs in both the A- and P-sites. Remarkably, all eleven modified nucleotides locate within this slice and are thus densely packed around the center of the ribosome. The following modified domain IV nucleotides are involved in contact with the 16 S rRNA of the small ribosomal subunit: {Psi}1932 (E. coli 1911), mU1936 (E. coli 1915), {Psi}1938 (E. coli 1917), and Cm1941 (E. coli 1920); these are all located in helix 69 of 23 S rRNA that forms contact to helices 44 and 45 of 16 S rRNA, whereas mC1983 (E. coli 1962) is at the basis of helix 71 in 23 S rRNA and contacts 16 S rRNA at helix 44. The three first modifications are highly conserved as either {Psi} or m3{Psi} among the domains of life (34, 46), but the high resolution structure of the E. coli ribosome (23) does not implicate a function of these modifications. The domain IV-methylated nucleotides also contact tRNA. mU1936 (E. coli 1915) interacts with the D-stem of tRNA in the A-site, whereas the methylated C1963 (E. coli 1942) is part of a conserved loop and contacts the acceptor stem of tRNA in the A-site. mU1960 (E. coli 1939) is also a part of the latter loop, but this nucleotide is not in direct contact with the A-site tRNA.

Three of the four modified domain V nucleotides are implicated in tRNA associations. Gm2262 (E. coli 2251) is part of the P-loop of 23 S rRNA and interacts with the tRNA acceptor tails in the ribosomal P-site. Um2563 (E. coli 2552) is the first nucleotide in the A-loop, which contacts aminoacyl-tRNA located in the A-site. The structural data indicate that it is not Um2563, but the subsequent G2564 (E. coli 2553), that contacts the A-site tRNA acceptor tail. Data from the large ribosomal subunit of H. marismortui suggests that ribose methylation of U2563 (E. coli 2552) is required for the A-loop to adapt the correct structure for interaction with neighboring nucleotides (7, 20). Finally, {Psi}2616 (E. coli 2605) is located in 23 S rRNA helix 93 that interact with the CCA-tail of P-site tRNA. A {Psi} at this position in the large ribosomal subunit RNA is a characteristic feature for eubacteria/chloroplasts, whereas the subsequent position is a {Psi} in eukaryotes (34, 46). The only methylated nucleotide in T. thermophilus not obviously involved in an RNA-RNA intermolecular contact was the base-methylated A-nucleotide in domain V at position 2514 (E. coli m2A2503). This nucleotide, which is also methylated in E. coli and B. megaterium as mentioned above, lines the start of the exit tunnel for the nascent polypeptide chain. We have no indications toward the precise function of this nucleotide, but hypermethylation of A2503 in E. coli generates resistance against a number of antibiotics (47).

In conclusion, our results show that most of the T. thermophilus 23 S rRNA post-transcriptionally modified nucleotides are directly or indirectly involved in intermolecular RNA-RNA bridges. It is, of course, tempting to speculate that some of the numerous post-transcriptional modifications in tRNA are required for interactions with rRNA in the ribosome. Such a role is found for 4-thiouridine (position 34) of E. coli tRNALys in contact with 16 S rRNA (48); this tRNA was used for A-site binding in the x-ray structure of the T. thermophilus ribosome (49). Thus, one central function post-transcriptional RNA modifications is probably modulation of intermolecular RNA-RNA contacts. An investigation of the T. thermophilus 16 S rRNA modification status will shed more light on the general involvement of rRNA modifications in intermolecular RNA-RNA contacts.

Note Added in Proof—An exhaustive map of T. thermophilus 16 S rRNA modifications was recently published (Guymon, R., Pomerantz, S. C., Crain, P. F., and McCloskey, J. A. (2006) Biochemistry 45, 4888-4899


   FOOTNOTES
 
* This work was supported by the Danish Biotechnology Instrument Centre and the Danish Natural Science Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Current address: Dept. of Forensic Medicine, University of Copenhagen, 2100 Copenhagen, Denmark. Back

2 To whom correspondence should be addressed. Tel.: 45-65-502-414; Fax: 45-659-327-81; E-mail: f.kir{at}bmb.sdu.dk.

3 The abbreviations used are: {Psi}, pseudouridine; MS, mass spectrometry; MS/MS, tandem mass spectrometry; MS3, third stage mass spectrometry; MALDI, matrix-assisted laser desorption/ionization; TEAA, triethylammonium acetate; TOF, time-of-flight; Cm, 2'-O-methylcytidine; Gm, 2'-O-methylguanosine; Um, 2'-O-methyluridine; m2A, 2-methyladenosine; mC, base-methylated cytidine; mA, base-methylated adenosine; mU, base-methylated uridine; Me, methyl; CMCT, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate; m3{Psi}, 3-methylpseudouridine; m1G, 1-methylguanosine; m5C, 5-methylcytidine; m5U, 5-methyluridine. Back

4 B. T. Porse and F. Kirpekar, unpublished observation. Back


   ACKNOWLEDGMENTS
 
T. thermophilus cells were a kind gift from V. Ramakrishnan. Erling Knudsen provided excellent technical assistance with bacterial growth.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Sirum-Connolly, K., Peltier, J. M., Crain, P. F., McCloskey, J. A., and Mason, T. L. (1995) Biochimie (Paris) 77, 30-39
  2. Andersen, T. E., Porse, B. T., and Kirpekar, F. (2004) RNA (N. Y.) 10, 907-913
  3. Rozenski, J., Crain, P. F., and McCloskey, J. A. (1999) Nucleic Acids Res. 27, 196-197[Abstract/Free Full Text]
  4. Maden, B. E. F. (1990) Progress Nucleic Acids Res. 39, 241-301
  5. Treede, I., Jacobsen, L., Kirpekar, F., Vester, B., Weitnauer, G., Douthwaite, S., and Bechthold, A. (2003) Mol. Microbiol. 49, 309-318[CrossRef][Medline] [Order article via Infotrieve]
  6. Skinner, R. H., and Cundliffe, E. (1982) J. General Microbiol. 128, 2411-2416
  7. Hansen, M. A., Kirpekar, F., Ritterbusch, W., and Vester, B. (2002) RNA (N. Y.) 8, 202-213
  8. Kirpekar, F., Hansen, L. H., Rasmussen, A., Poehlsgaard, J., and Vester, B. (2005) J. Mol. Biol. 348, 563-573[CrossRef][Medline] [Order article via Infotrieve]
  9. Cunningham, P. R., Richard, R. B., Weitzmann, C. J., Nurse, K., and Ofengand, J. (1991) Biochimie (Paris) 73, 789-796
  10. Green, R., and Noller, H. F. (1996) RNA (N. Y.) 2, 1011-1021
  11. Green, R., and Noller, H. F. (1999) Biochemistry 38, 1772-1779[CrossRef][Medline] [Order article via Infotrieve]
  12. Caldas, T., Binet, E., Bouloc, P., and Richarme, G. (2000) Biochem. Biophys. Res. Commun. 271, 714-718[CrossRef][Medline] [Order article via Infotrieve]
  13. Gustafsson, C., and Persson, B. (1998) J. Bacteriol. 180, 359-365[Abstract/Free Full Text]
  14. Raychaudhuri, S., Conrad, J., Hall, B. G., and Ofengand, J. (1998) RNA (N. Y.) 4, 1407-1417
  15. Gutgsell, N. S., Del Campo, M., Raychaudhuri, S., and Ofengand, J. (2001) RNA (N. Y.) 7, 990-998
  16. Liu, M., Novotny, G. W., and Douthwaite, S. (2004) RNA (N. Y.) 10, 1713-1720
  17. King, T. H., Liu, B., McCully, R. R., and Fournier, M. J. (2003) Mol. Cell 11, 425-435[CrossRef][Medline] [Order article via Infotrieve]
  18. Lowe, T. M., and Eddy, S. R. (1999) Science 283, 1168-1171[Abstract/Free Full Text]
  19. Yusupov, M. M., Yusupova, G. Z., Baucom, A., Lieberman, K., Earnest, T. N., Cate, J. H. D., and Noller, H. F. (2001) Science 292, 883-896[Abstract/Free Full Text]
  20. Ban, N., Nissen, P., Hansen, J., Moore, P. B., and Steitz, T. A. (2000) Science 289, 905-920[Abstract/Free Full Text]
  21. Clemons, W. M., May, J. L. C., Wimberley, B. T., McCutcheon, J. P., Capel, M. S., and Ramakrishnan, V. (1999) Nature 400, 833-840[CrossRef][Medline] [Order article via Infotrieve]
  22. Schlünzen, F., Zarivach, R., Bashan, A., Tocilj, A., Albrecht, R., Yonath, A., and Franceschi, F. (2001) Nature 413, 814-821[CrossRef][Medline] [Order article via Infotrieve]
  23. Schuwirth, B. S., Borovinskaya, M. A., Hau, C. W., Zhang, W., Vila-Sanjurjo, A., Holton, J. M., and Cate, J. H. D. (2005) Science 310, 827-834[Abstract/Free Full Text]
  24. Bakin, A., and Ofengand, J. (1993) Biochemistry 32, 9754-9762[CrossRef][Medline] [Order article via Infotrieve]
  25. Gogoya, Z. V., Yusupov, M. M., and Spirina, T. N. (1986) Mol. Biol. 20, 415-421
  26. Kirpekar, F., Douthwaite, S., and Roepstorff, P. (2000) RNA (N. Y.) 6, 296-306
  27. Mengel-Jorgensen, J., and Kirpekar, F. (2002) Nucleic Acids Res. 30, e135[Abstract/Free Full Text]
  28. Vogel, D. W., Hartmann, R. K., Kröger, B., Ulbrich, N., and Erdmann, V. A. (1987) Biochem. Int. 14, 167-175
  29. Hopfl, P., Ulbrich, N., Hartmann, R. K., Ludwig, W., and Schleifer, K. H. (1988) Nucleic Acids Res. 16, 9043[Free Full Text]
  30. Koradi, R., Billeter, M., and Wüthrich, K. (1996) J. Mol. Graphics 14, 51-55[CrossRef][Medline] [Order article via Infotrieve]
  31. Pomerantz, S. C., Kowalak, J. A., and McCloskey, J. A. (1993) J. Am. Soc. Mass Spectrom. 4, 204-209[CrossRef]
  32. McLuckey, S. A., Van Berkel, G. J., and Glish, G. L. (1992) J. Am. Soc. Mass Spectrom. 3, 60-70[CrossRef]
  33. Baer, R. J., and Dubin, D. T. (1981) Nucleic Acids Res. 9, 323-337[Abstract/Free Full Text]
  34. Del Campo, M., Recinos, C., Yanez, G., Pomerantz, S. C., Guymon, R., Crain, P. F., McCloskey, J. A., and Ofengand, J. (2005) RNA (N. Y.) 11, 210-219
  35. Liu, M., and Douthwaite, S. (2002) Mol. Microbiol. 44, 195-204[CrossRef][Medline] [Order article via Infotrieve]
  36. Gaspin, C., Cavaillé, J., Erauso, G., and Bachellerie, J.-P. (2000) J. Mol. Biol. 297, 895-906[CrossRef][Medline] [Order article via Infotrieve]
  37. Omer, A. D., Lowe, T. M., Russell, A. G., Ebhardt, H., Eddy, S. R., and Dennis, P. P. (2000) Science 288, 517-522[Abstract/Free Full Text]
  38. Barneche, F., Gaspin, C., Guyot, R., and Echeverría, M. (2001) J. Mol. Biol. 311, 57-73[CrossRef][Medline] [Order article via Infotrieve]
  39. Noon, K. R., Bruenger, E., and McCloskey, J. A. (1998) J. Bacteriol. 180, 2883-2888[Abstract/Free Full Text]
  40. Agris, P. F., Koh, P., and Söll, D. (1973) Arch. Biochem. Biophys. 154, 277-282[CrossRef][Medline] [Order article via Infotrieve]
  41. McCloskey, J. A., Graham, D. E., Zhou, S., Crain, P. F., Ibba, M., Konisky, J., Söll, D., and Olsen, G. J. (2001) Nucleic Acids Res. 29, 4699-4706[Abstract/Free Full Text]
  42. Cummins, L. L., Owens, S. R., Risen, L. M., Lesnik, E. A., Freier, S. M., McGee, D., Guinosso, C. J., and Cook, P. D. (1995) Nucleic Acids Res. 23, 2019-2024[Medline] [Order article via Infotrieve]
  43. Sengupta, R., Vainauskas, S., Yarian, C., Sochacka, E., Malkiewicz, A., Guenther, R. H., Koshlap, K. M., and Agris, P. F. (2000) Nucleic Acids Res. 28, 1374-1380[Abstract/Free Full Text]
  44. Ashraf, S. S., Guenther, R. H., Ansari, G., Malkiewicz, A., Sochacka, E., and Agris, P. F. (2000) Cell. Biochem. Biophys. 33, 241-252[CrossRef][Medline] [Order article via Infotrieve]
  45. Meroueh, M., Grohar, P. J., Qui, J., SantaLucia, J., Jr., Scaringe, S. A., and Chow, C. S. (2000) Nucleic Acids Res. 28, 2075-2083[Abstract/Free Full Text]
  46. Ofengand, J., and Bakin, A. (1997) J. Mol. Biol. 266, 246-268[CrossRef][Medline] [Order article via Infotrieve]
  47. Kehrenberg, C., Schwarz, S., Jacobsen, L., Hansen, L. H., and Vester, B. (2005) Mol. Microbiol. 57, 1064-1073[CrossRef][Medline] [Order article via Infotrieve]
  48. Ashraf, S. S., Sochacka, E., Cain, R., Guenther, R., Malkiewicz, A., and Agris, P. F. (1999) RNA (N. Y.) 5, 188-194
  49. Cate, J. H., Yusupov, M. M., Yusupova, G. Z., Earnest, T. N., and Noller, H. F. (1999) Science 285, 2095-2104[Abstract/Free Full Text]
  50. Aspegren, A., Hinas, A., Larsson, P., Larsson, A., and Söderbom, F. (2004) Nucleic Acids Res. 32, 4646-4656[Abstract/Free Full Text]
  51. Brown, J. W. S., Clark, G. P., Leader, D. J., Simpson, C. G., and Lowe, T. (2001) RNA (N. Y.) 7, 1817-1832
  52. Higa, S., Maeda, N., Kenmochi, N., and Tanaka, T. (2002) Biochem. Biophys. Res. Commun. 297, 1344-1349[CrossRef][Medline] [Order article via Infotrieve]
  53. Huang, Z.-P., Zhou, H., He, H.-L., Chen, C.-L., Liang, D., and Qu, L.-H. (2005) RNA (N. Y.) 11, 1303-1316
  54. Lestrade, L., and Weber, M. (2006) Nucleic Acids Res. 34, D158-D162[Abstract/Free Full Text]
  55. Liang, X.-H., Uliel, S., Hury, A., Barth, S., Doniger, T., Unger, R., and Michaeli, S. (2005) RNA (N. Y.) 11, 619-645
  56. Massenet, S., Ansmant, I., Motorin, Y., and Branlant, C. (1999) FEBS Lett. 462, 94-100[CrossRef][Medline] [Order article via Infotrieve]
  57. Samarsky, D. A., and Fournier, M. J. (1999) Nucleic Acids Res. 27, 161-164[Abstract/Free Full Text]
  58. Yang, C.-Y., Zhou, H., Luo, J., and Qu, L.-H. (2005) Biochem. Biophys. Res. Commun. 328, 1224-1231[CrossRef][Medline] [Order article via Infotrieve]

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