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Post-transcriptional modifications in the small subunit

ribosomal RNA from Thermotoga maritima, including
presence of a novel modified cytidine
REBECCA GUYMON,1,3 STEVEN C. POMERANTZ,1,4 J. NICHOLAS ISON,1 PAMELA F. CRAIN,1

and JAMES A. MCCLOSKEY1,2
1
Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112, USA
2
Department of Biochemistry, University of Utah, Salt Lake City, Utah 84112, USA
ABSTRACT
Post-transcriptional modifications of RNA are nearly ubiquitous in the principal RNAs involved in translation. However, in the
case of rRNA the functional roles of modification are far less established than for tRNA, and are subject to less knowledge in terms
of specific nucleoside identities and their sequence locations. Post-transcriptional modifications have been studied in the SSU
rRNA from Thermotoga maritima (optimal growth 80°C), one of the most deeply branched organisms in the Eubacterial
phylogenetic tree. A total of 10 different modified nucleosides were found, the greatest number reported for bacterial SSU rRNA,
occupying a net of ;14 sequence sites, compared with a similar number of sites recently reported for Thermus thermophilus and
11 for Escherichia coli. The relatively large number of modifications in Thermotoga offers modest support for the notion that
thermophile rRNAs are more extensively modified than those from mesophiles. Seven of the Thermotoga modified sites are
identical (location and identity) to those in E. coli. An unusual derivative of cytidine was found, designated N-330 (Mr 330.117),
and was sequenced to position 1404 in the decoding region of the rRNA. It was unexpectedly found to be identical to an earlier
reported nucleoside of unknown structure at the same location in the SSU RNA of the archaeal mesophile Haloferax volcanii.
Keywords: post-transcriptional modification; Thermotoga maritima; Haloferax volcanii; small ribosomal subunit RNA;
liquid chromatography–mass spectrometry; electrospray ionization
INTRODUCTION by Decatur and Fournier 2002), in contrast to tRNA, in

which a number of modification functions are slowly being
Relatively little is known concerning the specific roles of
unraveled (Agris 2004). In the case of rRNA, the influence
post-transcriptional modifications in rRNA (summarized
of nucleotide modification on ribosome assembly and
maturation (Bachellerie and Cavaillé 1998; Maden 1998;
3
Present addresses: ARUP Laboratories, 500 Chipeta Way, Salt Lake City, Mason 1998) is perhaps the best documented (Vaughan
UT 84108, USA. 4Centocor, 145 King of Prussia Rd., Radnor, PA 19087, USA. et al. 1967; Cunningham et al. 1990, 1991; Rydén-Aulin et al.
Reprint requests to: James A. McCloskey, University of Utah, 30 S. 1993; Sirum-Connolly and Mason 1993; Sirum-Connolly
2000 East, Rm. 311A, Salt Lake City, UT 84112-5820, USA; e-mail:
[email protected]; fax: (801) 581-7087. et al. 1995; Green and Noller 1996), but mechanistic details
Abbreviations: LC/ESI-MS, combined liquid chromatography–electro- at the molecular level have been scant. There is strong
spray ionization mass spectrometry; C, pseudouridine; m3U, 3-methylur- circumstantial evidence in the archaeal thermophiles that
idine; Cm, 29-O-methylcytidine; m4Cm, N4,O-29-dimethylcytidine; m5C,
5-methylcytidine; m2G, N2-methylguanosine; m7G, 7-methylguanosine; ribose methylation at O29 provides a significant mechanism
Am, 29-O-methyladenosine; m26A, N6,N6-dimethyladenosine; MS/MS, for thermal stabilization of rRNA (Noon et al. 1998) and
tandem mass spectrometry; M3 , molecular ion having three negative of archaeal tRNA (Kowalak et al. 1994; Noon et al.
charges, corresponding to loss of three protons from the neutral molecule
M, i.e., (M 3H)3 (analogous definitions for other charge states); MH+, 2003), primarily through reduced flexibility resulting from
protonated molecule ion; BH+2, protonated base ion in which B is the base strong thermodynamic enforcement of the C39-endo sugar
fragment attached to ribose and BH corresponds to the neutral base; CID, conformation (Kawai et al. 1992). Also, from the standpoint
collision-induced dissociation.
Article published online ahead of print. Article and publication date are of function, selective methylation of rRNA nucleotides
at http://www.rnajournal.org/cgi/doi/10.1261/rna.361607. provides, in some instances, an effective mechanism for
396 RNA (2007), 13:396–403. Published by Cold Spring Harbor Laboratory Press. Copyright Ó 2007 RNA Society.
rna3616 Guymon et al. ARTICLE RA

Modifications in Thermotoga 16S rRNA
conferring resistance to antibiotics that target the bacterial Identification and sequence location of modified
ribosome (Douthwaite et al. 2005). residues in T. maritima 16S rRNA
Many of the structures and sequence locations of rRNA
RNA modifications represented by changes in mass from
modifications are relatively well conserved (Maden 1990;
the parent nucleotide were mapped. The locations of
Rozenski and McCloskey 2005) and exhibit distinct differ-
pseudouridines, which are mass silent, were not measured
ences among the three principal evolutionary domains,
beyond the estimation of 3.8 net residues in the molecule.
Archaea, Bacteria, and Eukarya. The complete modification
Chromatographic separation of RNase T1 digestion prod-
map for SSU rRNA of the bacterial thermophile Thermus
ucts of T. maritima SSU rRNA is shown in Figure 1, with
thermophilus was recently completed (Guymon et al. 2006)
the corresponding mass data for modified oligonucleotides
and found to exhibit a notably lower level of modification
given in Table 1. A section of the RNase U2 chromatogram
than that of the archaeal thermophile Sulfolobus solfataricus
in which modified oligonucleotides were eluted is shown in
(Noon et al. 1998) growing in the same temperature range
Supplemental Figure S2.
(70°C–75°C), and a pattern of modification (nucleoside
To a certain extent the procedures used for identification
structures and sequence locations) unexpectedly similar
and sequence placement of modified residues are analogous
to that in the mesophile Escherichia coli. For a tabulation
to those recently used and described in a study of
of E. coli rRNA modifications and leading references see
T. thermophilus SSU rRNA (Guymon et al. 2006). Therefore,
http://medlib.med.utah.edu/RNAmods/.
descriptions of results for seven modified residues in the
We have examined the SSU rRNA modifications in a
present work can be found in the Supplemental Data section.
second bacterial thermophile, Thermotoga maritima (opti-
These are: m7G-527 (Supplemental Fig. S3), m2G-966 and
mal growth at 80°C) (Huber et al. 1986), an organism
m5C-967 (Supplemental Fig. S4), m2G-1051 (Supplemental
placed by small subunit ribosomal RNA (SSU rRNA)
Table S2), m4Cm-1402 (Supplemental Fig. S5), Cm-1409
phylogeny as one of the most deeply rooted organisms in
(Supplemental Figs. S6,S7), and 1518-m62Am62A-1519
the Eubacterial tree. Analysis of the complete Thermotoga
placement. Results for N-330–1404, m3U-1498, and Am-
genome sequence (Nelson et al. 1999) as well as earlier
1499 follow below.
data (Aravind et al. 1998) led to conclusions that extensive
lateral gene transfer between T. maritima and Archaea have
N-330–1404
occurred, reflected in the unusually high percentage (24%)
of genes in T. maritima that are similar to those in Archaea. The presence of a novel structurally unknown nucleoside of
Examination of T. maritima SSU rRNA in the present study Mr 330 at position 1404 is indicated from the following
was intended in part to establish whether its modifications four lines of evidence. (1) The sequencing spectrum of
are to any extent archaeal in nature, and thus reflecting T1 oligonucleotide Mr 1393 (Supplemental Fig. S5) can be
possible horizontal transfer of RNA modification enzymes. assigned only if a unique nucleotide residue mass of 392
Further, this study serves to extend the knowledge base of (N-330p minus H2O) is used in the third nucleotide
bacterial SSU rRNA modification patterns (Rozenski and position. (2) The base– (m/z 197) and N > p– (m/z 391)
McCloskey 2005; Guymon et al. 2006) beyond E. coli and monomer fragment ion signals (Supplemental Table S1)
T. thermophilus, the only bacteria for which complete SSU were assigned from the data in Supplemental Figure S5 and
modification maps are available, and beyond organisms for time aligned as shown in Supplemental Figure S8. Ions
which RNase T1 catalog data were reported (Rozenski and
McCloskey 2005) in which modifications were sometimes
reported to occur, often without chemical identity of the
modified nucleoside or firm knowledge of its sequence
location in the 16S RNA molecule.
RESULTS
Modified nucleoside content of T. maritima 16S rRNA

Identities and approximate numbers of modified nucleo-
sides, determined by LC/ESI-MS analysis of total nucleo-
side digests of 16S rRNA (Supplemental Fig. S1, at http://
library.med.utah.edu/mccloskey) are: C (3.8 residues),
m3U (1.0), m4Cm (1.2), m5C (0.73), Cm (0.82), unknown
FIGURE 1. HPLC separation of RNase T1 digestion products of
nucleoside N-330 (UV molar absorptivity not known, so T. maritima 16S rRNA, annotated to show modified oligonucleotides
stoichiometry not estimated), m2G (2.3), m7G (1.1), Am determined from mass shifts in conjunction with data in Table 1. UV
(trace level to 1.3; see text), and m 62A (2.1). absorbance detection at 260 nm.
www.rnajournal.org 397
398
Guymon et al.
RNA, Vol. 13, No. 3

TABLE 1. Modified nucleosides in RNases T1 and U2 hydrolysis products from Thermotoga maritima 16S rRNA
RNase T1 RNase U2
Mr meas. Monomer ions, Mr meas. Monomer ions,

Modification Sequencea (error)b m/zc Sequencea (error)b m/zc
m7G-527 525-CCm7GCGpd-529 1637.29 (0.05) 164 525-CCm7GCGG>p-530 1964.31 (0.03)

m2G-966 964-AUm2Gp-966 1012.16 (0.01) 164, 358, 376 965-Um2Gm5CUA>p-969 1619.26 (0.04) 164, 358, 376, 124, 336
m5C-967 967-m5CUAAGp-971 1646.25 (0.01) 124, 336
m2G-1051 1051-m2G>p-1051 359.03 (0.03) 164 1049-UGm2GUG>pd-1053 1661.25 (0.04) 164, 358
m4Cm-1402, 1402-m4CmCN-330Gpd-1405 1393.28 (0.01) 124, 350, 197 1399-CCGm4CmCN-330GUCA>p-1408 3270.53 (0.06)
N-330–1404 (N-330 base),
391 (N-330>p)
Cm-1409 1406-UCACmGpd-1410 1622.24 (0.01) 336, 398 pCm>p Mass not found through tracking
m3U-1498, 1498-m3UAmACAAGpd-1504 2318.43 (0.07) 125, 319, 337, 1495-UCGm3UA>p-1499 1605.26 (0.06) 125, 319, 337, 342
Am-1499 225, 342 1495-UCGm3UAm>p-1499 1620.16 (0.17) avg.
m 62A-1518, 1518-m 62Am 62AGp-1520 1077.23 (0.01) 162, 356, 374 Mass not found through tracking
1519
a
E. coli sequence numbering used throughout.
b
Mass was experimentally measured; error is found minus calculated mass for the modified oligonucleotide shown.
c
See also Supplemental Table S1.
d
Sequenced by mass spectrometry.
corresponding to the modified residue m4Cm, whose ion, m/z 148, was observed). This methylation pattern
presence is indicated from the total nucleoside analysis was supported by the finding in the U2 digest of the
data in Supplemental Figure S1, were also time aligned. The 39-truncated version of this sequence but having an
exact coelution pattern of ions from N-330 and m4Cm is extension of three nucleotides on the 59 end, Mr 1620
shown in Supplemental Figure S8, and supports the (see Table 1). However, the assignment of Am-1499 in
presence of both of those nucleosides in the T1 oligonu- these RNase products appeared to be at odds with the
cleotide Mr 1393. The characteristic bacterial nucleoside absence of the methylribose cyclic phosphate ion (m/z 225)
m4Cm serves as an internal marker for position 1402 in the spectrum, usually a strong indicator of presence of
(Rozenski and McCloskey 2005) and correlates with the a ribose-methylated nucleotide (Phillips and McCloskey
full oligonucleotide sequence derived by mass spectrometry 1993). To address this point the product ion mass spectrum
in Supplemental Figure S5. (3) The presence of a base- of chemically synthesized m3UAmACAAGp was acquired.
modified unknown nucleoside of Mr 330 (MH+, m/z 331; It appeared essentially identical (data not shown) to the
BH +2, m/z 199; lmax 270) eluting at 3.85 min in the total mass spectrum displayed in Figure 2, including the absence
nucleoside digest of Thermotoga 16S rRNA, Supplemental of ion m/z 225. The sequence of the Am-containing region
Figure S1. (4) Modification of the universal C-1404 was is therefore assigned as shown in Table 1. The occurrence
also reported in several other bacterial SSU rRNAs of Am is further supported by its presence in the total
(although not in E. coli) (Rozenski and McCloskey 2005), nucleoside digest (Fig. S1), with MH+ and BH +2 ions
but not the identity of the modification. appearing at the expected retention time (Pomerantz
and McCloskey 1990). Interestingly, a second culture of
m3U-1498 and Am-1499 T. maritima showed an otherwise very similar overall
modification data set, but with the 1495–1499 fragment
Assignment of the unusual tandem methylation sites 1498
occurring primarily as Mr 1605.2 (see Table 1), i.e., with
and 1499 was made primarily through the T1 product Mr
one fewer methyl group. We interpret this finding, taken in
2318 (Table 1). The experimental mass 2318.43, using the
conjunction with variable amounts of Am found in
corresponding gene sequence to calculate allowable RNA
nucleoside total digests from two different T. maritima
sequences, solely designates the unique sequence segment
cultures, as an indication that the level of Am-1499 is both
as shown in Table 1 corresponding to nucleotides 1498–
substoichiometric and variable in the rRNA. To our
1504 with two methyl groups. The sequence mass spectrum
knowledge, this is the first report of Am in RNA from
of this T1 fragment (Fig. 2) requires, through the presence
bacteria; however, using LC/ESI-MS we have observed Am
of a clear y1–y5 ion series (McLuckey et al. 1992), that the
in unfractionated tRNA from E. coli MRE 600 (F. Qiu and
methyl groups be confined to the first two nucleotides. The
J. McCloskey, unpubl., 1998).
abundant methyluracil base ion m/z 125, in conjunction
with the total nucleoside census (Supplemental Fig. S1) then
implies m3U in position 1498 and Am, the only methylated 39-Terminal RNase T1 fragment from T. maritima
A in the rRNA, at position 1499. (No methyladenine base 16S rRNA
The terminal fragment is a 15-mer, expected to contain the
anti-Shine–Dalgarno mRNA-binding sequence. Because of
the recent unusual finding of adjacent pseudouridines at
1540 and 1541 in the 39-tail of T. thermophilus (Guymon
et al. 2006), the Thermotoga 39-terminal fragment was sub-
jected to a C detection protocol (Pomerantz and McCloskey
2005) using LC/ESI-MS/MS. No C was detected. The ter-
minal fragment was mass measured as 4591.7 (4591.60
calc.), and MS-sequenced as AUCACCUCCUUUCUAOH
(Supplemental Fig. S9), thus revealing the molecular
terminus as AOH-1545 (E. coli numbering) in the pro-
cessed 16S molecule.
Presence of nucleoside N-330 in Haloferax volcanii

16S rRNA
FIGURE 2. Product ion mass spectrum from RNase T1 fragment Mr The discovery of a new modified nucleoside in the
2318, showing m3U-1498 adjacent to Am-1499. For clarity, only peaks
used for sequence assignment are annotated. Sequence coverage from
Thermotoga SSU at the same position previously described
the four sequence ion series (a–B, d–H2O, w, and y) (McLuckey et al. as containing a modified cytidine in H. volcanii 16S rRNA
1992) is shown by horizontal bars. (Gupta et al. 1983) prompted a revisit of our previously
Guymon et al.
published study of the Haloferax SSU RNA modifications conserved in Bacteria than in the Archaea and Eukarya. For
(Kowalak et al. 2000). When the ion chromatograms for example, modification levels in Archaeal 16S RNA range
the diagnostic MH+ and BH +2 ions for N-330 nucleoside from five residues in H. volcanii (Gupta et al. 1983) to z38
(m/z 331 and 199, respectively) were extracted from our in S. solfataricus (Noon et al. 1998). Modification at z8–11
Haloferax total nucleoside digest data, their presence was SSU RNA sequence sites appears to be most common in
confirmed by the appearance of these two ions in a peak bacteria (E. coli has 11) while the elevation by about 30% of
exactly coeluting at 3.6 min (Fig. 3). If N-330 is, in fact, the modification levels in the bacterial thermophiles (versus
‘‘C*’’ nucleotide previously reported, the calculated mass of mesophiles) supports the conclusion that post-transcrip-
the predicted Haloferax N-330-containing T1 oligonucleo- tional modification in general serves to support structural
tide (CCC*Gp) would be Mr 1365.7. Reconstructed ion stabilization of RNA (Sampson and Uhlenbeck 1988;
chromatograms were therefore generated using the M Derrick and Horowitz 1993; Kowalak et al. 1994). The
and M2 ions (m/z 1364.7 and m/z 681.8, respectively), and presence of 11 modifications was recently reported for
for the B ion for the base of N-330 (m/z 197). As shown in T. thermophilus 23S rRNA (Mengel-Jorgensen et al. 2006),
Supplemental Figure S10, each of the three ions forms a compared with z23 in E. coli LSU RNA. This relative
peak eluting at 16.0 min, indicating N-330 to be the concentration is significantly lower than expected, based on
modified C* nucleotide found by Woese and colleagues our finding of 14 sites in the Thermus 16S RNA. However,
(Gupta et al. 1983). the methodology used (Mengel-Jorgensen et al. 2006) was
not designed to provide a complete census of modifications
that required placement. As in the case of T. thermophilus
DISCUSSION
(Guymon et al. 2006), rRNA modification levels in Ther-
motoga are characteristically much lower than in the
Modification identities and levels in T. maritima
Archaeal thermophiles growing at about the same temper-
SSU RNA
ature (Noon et al. 1998), with considerably less reliance on
The finding of 10 different modified nucleosides at a net ribose O-29 methylation as a major means of structural
occupancy level of z14 sequence sites marks T. maritima, stabilization. The finding of a net 3.8 residues of C in
and similarly T. thermophilus (Guymon et al. 2006), as the Thermotoga SSU RNA is notable in that C has been shown
most extensively modified bacterial SSU RNAs presently to play a strong, although often overlooked role in RNA
known. Examination of a number of catalogued RNase T1 stabilization (Davis 1995, 1998), and the level in Thermo-
modification maps, although by their nature less complete toga appears to be the highest concentration of C reported
than the full modification maps from E. coli (leading in bacterial SSU RNAs (Ofengand and Rudd 2000).
citations in ref. Bakin et al. 1994; see also http://medlib. The availability of three bacterial SSU RNA modification
med.utah.edu/RNAmods), and T. thermophilus (Guymon maps, E. coli (Brosius et al. 1978), Thermus (Guymon et al.
et al. 2006) SSU RNAs, suggests that the levels and 2006), and now Thermotoga, when coupled with informa-
identities of modifications are perhaps narrower and more tion from T1 catalogs (Rozenski and McCloskey 2005)
permits an estimation of the most highly conserved
modification sites that are unique to bacteria, even though
most of the T1 catalog data identify sites but not chemical
structures of modifications. In bacteria these sites are
C*-967 in h31 and U*-1498 in the center of the decoding
region in h44. An exception to the bacterial uniqueness of a
modified C-967 appears to occur in the archaeon Thermo-
proteus tenax (Woese et al. 1984). The position 967
modification is reported to be m5C in all four cases in
which the nucleoside structure has been established (E. coli,
Thermus, Thermotoga, and Proteus). The adjacent modifi-
cation m2G-966, implicated in P-site tRNA binding (von
Ahsen and Noller 1995; Korostelev et al. 2006), represents
an essentially universal SSU modification site in all phylo-
genetic domains (Rozenski and McCloskey 2005) and is
represented by an interesting diversity of modified nucle-
FIGURE 3. Demonstration of presence of modified nucleoside N-330 oside structures in Archaea and Eukarya (Kowalak et al.
in a total nucleoside digest of H. volcanii 16S rRNA (from previously 2000). The identity of U*-1498 has been established
acquired data) (Kowalak et al. 2000), detected from reconstructed ion
chromatograms (RIC) for characteristic positively charged ions. (A)
specifically as m3U in the same four organisms. The
UV absorbance at 260 nm. (B) RIC for the MH+ ion of N-330 (m/z unusual dimethylcytidine m4Cm-1402 is unique to bacte-
331). (C) RIC for the protonated base fragment ion of N-330 (m/z 199). rial rRNA (Limbach et al. 1994), but this site appears to be
400 RNA, Vol. 13, No. 3

modified in fewer than half of the reported cases (Rozenski structure to be more complex than any of the nine known
and McCloskey 2005). In T. thermophilus the m4Cm modified cytidines in rRNA (Rozenski and McCloskey
residue serves to stabilize the third tRNA nucleotide by 2005). The accurate molecular mass of N-330 was mea-
H binding of its phosphate to the 4-methylamino moiety sured as 330.117 6 0.002 using a Micromass Q-Tof mass
(Korostelev et al. 2006). spectrometer. This value is consistent with an elemental
composition of C12H18N4O7 (calc. 330.1175), thus requir-
ing a side chain having one N atom, with three Ns
Modifications at the functional center of the ribosome
accounted for by the cytosine heterocycle. Unfortunately,
As deduced in earlier literature, modification sites in the exact chemical structure of N-330-1404 cannot be
bacterial SSU RNAs tend to occur, in three-dimensional further pursued due to laboratory closure and retirement
space, near the decoding center of the RNA (Brimacombe of the corresponding author.
et al. 1993; Decatur and Fournier 2002). Four of the 16S It is interesting to consider the possibility that the
modifications (m2G-966, m5C-1400, m4Cm-1402, and structures and distributions of post-transcriptionally mod-
m3U-1498) were determined by X-ray crystallography to ified nucleosides—the specific products of a sizable array of
support interaction between 16S RNA and the P-site codon RNA modification enzymes—could, in some instances, be
and anticodon stem–loop (Korostelev et al. 2006). These indicators of past horizontal transfer of DNA coding for
observations reflect the net importance of modification to modification enzymes. This notion is supported by the
efficient ribosomal function, as has been stated (Decatur sharp lines of phylogenetic demarcation of some modified
and Fournier 2002). The high level of modification in the nucleoside families (Limbach et al. 1994; Motorin and
upper portion of helix 44 in SSU RNA occurs at the Grosjean 1998). The possibility of lateral transfer was
interface with the LSU RNA, forming a cavity in which earlier raised (McCloskey et al. 2001) in light of the strong
translation occurs. These modifications in h44 include six clustering of the (otherwise bacterial) anticodon nucleoside
methyl groups each in Thermotoga and Thermus, opposite mnm5s2U in tRNAs from the Methanococci, a lineage of
an analogous concentration of modifications on the 23S methanogenic marine archaea. Interestingly, nucleoside
side of the Thermus LSU (Mengel-Jorgensen et al. 2006). N-330 is the only rRNA nucleoside occurring in Thermotoga
Mengel-Jorgensen et al. (2006) have concluded that the that is shared only by bacteria and archaea, all others (with
occurrence of modifications in the 23S RNA of Thermus the exception of m4Cm-1402, which is uniquely bacterial)
principally at the RNA–RNA interface suggests that they being found in all three phylogenetic domains. Nucleoside
play a role in modulating the RNA–RNA interface contact. N-330 might therefore be, in part, the product of RNA
Their conclusions are supported by the locations of modification enzyme genes transferred horizontally
modifications in 16S RNA from Thermotoga (present between Haloferax and Thermotoga. This speculation is
work), Thermus (Guymon et al. 2006), and a series of based on the report of extensive lateral transfer between
studies on E. coli (Brimacombe et al. 1993; Decatur and Thermotoga and archaea (Aravind et al. 1998; Logsdon and
Fournier 2002), showing that the ribosomal subunit inter- Faguy 1999), such that almost one-quarter of the Thermo-
face is intimately associated with post-transcriptional toga genome (24%) was found to be archaeal in nature
modifications. (Nelson et al. 1999). Work in the area of RNA modification
enzymes (Leung et al. 1998), however, while extensive in
some notable cases, such as the yeast tRNA modification
Modified nucleoside N-330–1404 in the decoding
enzymes (Johansson and Byström 2005) and the pseudo-
region of the RNA
uridine synthases (Ofengand and Fournier 1998), by its
Unknown N-330 is remarkable in two ways: first, in terms nature generally lags behind knowledge of modified nucle-
of its structural properties as inferred thus far; second, its oside structures (now numbering about 106; see http://
unexpected occurrence in another phylogenetic domain, medlib.med.utah.edu/RNAmods) and their phylogenetic
at the same location in the SSU RNA of the archaeal distributions. Pursuit of this possibility that N-330 is a
mesophile H. volcanii. Interestingly, C-1404 has been product of lateral gene transfer will require knowledge of
reported as modified but with unknown structure in the the chemical structure of N-330, and also of the sequences
RNase T1 SSU maps of five bacteria (Rozenski and of specific enzymes responsible for its formation.
McCloskey 2005), and two archaea, H. volcanii (Gupta
et al. 1983) and S. solfataricus (Woese et al. 1984). C-1404 MATERIALS AND METHODS
pairs with essentially universal G-1497 (Cannone et al.
Thermotoga maritima MSB8 cells were grown at 80°C at The
2002) near the top of h44. N-330 is implied to be a University of Georgia Bioexpression and Fermentation Facility,
derivative of cytidine by the corresponding gene sequence under the supervision of T.E. Davies, and RNA from this source
(Gupta et al. 1983). The molecular mass of 330 Da is was acquired in two ways. First, purified 16S rRNA was obtained
unique among all known modified nucleosides in RNA (see as a gift from V. Ramakrishnan (Department of Biochemistry,
http://medlib.med.utah.edu/RNAmods) and suggests its University of Utah; present address MRC Laboratory of Molecular
Guymon et al.
Biology, Cambridge, U.K.). Second, T. maritima 30S ribosomal Cannone, J.J., Subramanian, S., Schnare, M.N., Collett, J.R.,
subunits were obtained from the same source and 16S RNA was D’Souza, L.M., Du, Y., Feng, B., Lin, N., Madabusi, L.V.,
extracted from the 30S ribosomes with TRI Reagent (Chomczynski Muller, K.M., et al. 2002. The comparative RNA web (CRW) site:
An online database of comparative sequence and structure
and Sacchi 1987) following the manufacturer’s protocol. 16S
information for ribosomal, intron, and other RNAs. BMC Bio-
rRNAs (typically, 100 mg; 200 pmol) were digested totally to informatics 3: 2.
nucleosides with nuclease P1, phosphodiesterase I, and BAP Chomczynski, P. and Sacchi, N. 1987. Single-step method of RNA
(Crain 1990). Aliquots of RNA (5–100 pmol) were digested with isolation by acid guanidinium thiocyanate–phenol–chloroform
1000 units of RNase T1 (Ambion) in 10 mM Tris+ 1 mM EDTA, extraction. Anal. Biochem. 162: 156–159.
pH 7 for 30 min at 37°C or 45 min at 55°C. Alternatively, the Crain, P.F. 1990. Preparation and enzymatic hydrolysis of RNA and
RNA was digested with RNase U2 (Industrial Research Ltd.) in 20 DNA for mass spectrometry. Methods Enzymol. 193: 782–790.
Cunningham, P.R., Weitzmann, C.J., Nègre, D., Sinning, J.G.,
mM diammonium citrate (DAC), pH 5, 1 mM EDTA, or in 20 Frick, V., Nurse, K., and Ofengand, J. 1990. In vitro analysis of
mM DAC, pH 5, 1 mM EDTA, 8 M urea, with 5–10 units RNase the role of rRNA in protein synthesis: Site-specific mutation and
U2 for 15 min at 60°C, then an additional 5–10 units of enzyme methylation. In The ribosome: Structure, function, and evolution
was added and digestion continued for 15 min. Details of the mass (eds. W.E. Hill et al.), pp. 243–252. ASM Press, Washington, DC.
spectrometry methods used in this study can be found in the Cunningham, P.R., Richard, R.B., Weitzmann, C.J., Nurse, K., and
Supplemental Data section. Ofengand, J. 1991. The absence of modified nucleotides affects
both in vitro assembly and in vitro function of the 30S-ribosomal
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Post-transcriptional modifications in the small subunit ribosomal RNA

from Thermotoga maritima, including presence of a novel modified
cytidine
Rebecca Guymon, Steven C. Pomerantz, J. Nicholas Ison, et al.
RNA 2007 13: 396-403 originally published online January 25, 2007
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JOBNAME: RNA 13#3 2007 PAGE: 1 OUTPUT: Wednesday January 31 23:37:27 2007

Post-transcriptional modifications in the small subunit

REBECCA GUYMON,1,3 STEVEN C. POMERANTZ,1,4 J. NICHOLAS ISON,1 PAMELA F. CRAIN,1

INTRODUCTION by Decatur and Fournier 2002), in contrast to tRNA, in

rna3616 Guymon et al. ARTICLE RA

Modifications in Thermotoga 16S rRNA

Modified nucleoside content of T. maritima 16S rRNA

RNA, Vol. 13, No. 3

Mr meas. Monomer ions, Mr meas. Monomer ions,

m7G-527 525-CCm7GCGpd-529 1637.29 (0.05) 164 525-CCm7GCGG>p-530 1964.31 (0.03)

Modifications in Thermotoga 16S rRNA

Presence of nucleoside N-330 in Haloferax volcanii

400 RNA, Vol. 13, No. 3

Modifications in Thermotoga 16S rRNA

402 RNA, Vol. 13, No. 3

Modifications in Thermotoga 16S rRNA

Post-transcriptional modifications in the small subunit ribosomal RNA

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