THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 277, No. 11, Issue of March 15, pp. 8835–8840, 2002
Printed in U.S.A.
Characterization of the 23 S Ribosomal RNA m5U1939
Methyltransferase from Escherichia coli*
Received for publication, December 12, 2001
Published, JBC Papers in Press, January 4, 2002, DOI 10.1074/jbc.M111825200
Sanjay Agarwalla, James T. Kealey‡, Daniel V. Santi‡, and Robert M. Stroud§
From the Departments of Biochemistry and Biophysics and Pharmaceutical Chemistry, University of California
at San Francisco, San Francisco, California 94143-0448
An Escherichia coli open reading frame, ygcA, was
identified as a putative 23 S ribosomal RNA 5-methyluridine methyltransferase (Gustafsson, C., Reid, R.,
Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res.
24, 3756 –3762). We have cloned, expressed, and purified
the 50-kDa protein encoded by ygcA. The purified enzyme catalyzed the AdoMet-dependent methylation of
23 S rRNA but did not act upon 16 S rRNA or tRNA. A
high performance liquid chromatography-based nucleoside analysis identified the reaction product as
5-methyluridine. The enzyme specifically methylated
U1939 as determined by a nuclease protection assay and
by methylation assays using site-specific mutants of 23 S
rRNA. A 40-nucleotide 23 S rRNA fragment (nucleotide
1930 –1969) also served as an efficient substrate for the
enzyme. The apparent Km values for the 40-mer RNA
oligonucleotide and AdoMet were 3 and 26 !M, respectively, and the apparent kcat was 0.06 s"1. The enzyme
contains two equivalents of iron/monomer and has a
sequence motif similar to a motif found in iron-sulfur
proteins. We propose to name this gene rumA and accordingly name the protein product as RumA for RNA
uridine methyltransferase.
Most stable RNAs contain post-transcriptionally modified
nucleotides that are derivatives of the four common nucleotides
(1, 2). The enzymes that carry out these modifications are often
position-specific, and the specificity is largely determined by
the tertiary structure of the RNA (3– 6). Approximately 1% of a
bacterial genome is devoted to encoding RNA modification enzymes. Escherichia coli 16 S rRNA1 has a total of 11 modifications of which 10 are methylated nucleotides, whereas 23 S
rRNA has a total of 23 modifications of which 14 are methylated nucleotides (2, 7). Most rRNA modifications are clustered
near the functionally important regions of ribosome and are
thus speculated to have important functions.
For the 24 methylated nucleotides found in E. coli rRNA,
* This work was supported by the United States Public Health Service, National Institutes of Health Grant GM51232 (to R. M. S.). 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.
‡ Present address: Kosan Biosciences, Inc., 3832 Bay Center Place,
Hayward, CA 94545.
§ To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, University of California at San Francisco, San
Francisco, California 94143-0448. Tel.: 415-476-4224; Fax: 415-4761902; E-mail: [email protected].
1
The abbreviations used are: rRNA, ribosomal RNA; Me, methyl;
m5U, 5-methyluridine; ORF, open reading frame; AdoMet, S-adenosyl
methionine; HPLC, high performance liquid chromatography; ",
pseudouridine; MES, 4-morpholineethanesulfonic acid.
This paper is available on line at http://www.jbc.org
only six methyltransferases responsible for seven methylated
nucleotides have been characterized. In 16 S rRNA, Fmu
methylates the C5 of C967 (8, 9), RsmA or KsgA, a member of
the Erm family, catalyzes the exocyclic N6 dimethylation of
adenosine at positions 1518 and 1519 (10), and RsmC methylates the N2 of G1207 in 16 S rRNA (11). In 23 S rRNA, RrmA
methylates the N1 of G745 (12), RlmB methylates the 2!-Oribose of G2251 (13), and RrmJ methylates the 2!-O-ribose of
U2552 (14).
A common modification found in RNA is the substitution of
5-methyluridine (m5U) for uridine. Two m5Us have been identified at positions 747 and 1939 in E. coli 23 S rRNA. U747 is
located in the conserved loop of hairpin 35 in domain II of large
subunit rRNA. This loop lines the polypeptide exit channel and
makes tertiary contacts with domain V (15). U1939 is also
present in a conserved loop. In the crystal structure of the 70 S
ribosome in complex with tRNAs, the U1939 loop is seen protruding into the major groove of the A-site tRNA near the end
of the acceptor stem (16).
A highly conserved m5U is found at position 54 in tRNA, and
the modification is catalyzed by the enzyme, RUMT (also called
TrmA) (17–20). Sequence homology searches using the RUMT
sequence as probe identified two E. coli open reading frames
(ORFs), namely ybjF and ygcA, which were predicted to encode
m5U methyltransferases (21). This report also suggested that
the two ORFs would encode the enzymes responsible for the
modifications at positions 747 and 1939. In this study, we
describe the cloning of ygcA and the purification and characterization of its protein product. We have demonstrated that
the product of ygcA is the 23 S rRNA m5U1939 methyltransferase. In view of its established function, we propose to rename ygcA as rumA and the protein product as RumA.
EXPERIMENTAL PROCEDURES
Materials—Restriction enzymes were from New England Biolabs.
SP-Sepharose, phenyl-Sepharose, and [3H-Me]S-adenosyl methionine
(AdoMet) were purchased from Amersham Biosciences, Inc. Hydroxyapatite was obtained from Bio-Rad. DEAE-ion exchange paper, DE81,
was from Whatman. Plasmid p67YF0 was a gift from Prof. O. C.
Uhlenback (Department of Chemistry and Biochemistry, University of
Colorado, Boulder, CO), and plasmids pWK1 and pCW1 were gifts from
Prof. J. Ofengand (Department of Biochemistry and Molecular Biology,
School of Medicine, University of Miami, Miami, FL). DNA and RNA
oligonucleotides were purchased from Invitrogen and Dharmacon Research, Inc., respectively. Nuclease P1 and alkaline phosphatase were
from Roche Molecular Biochemicals. Nucleoside standards and other
chemicals were purchased from Sigma.
Cloning of rumA—The rumA (ygcA) ORF was amplified from E. coli
genomic DNA by PCR using the N-terminal primer 5!-TACTTGCCATGGCGCAATTCTACTCTG-3! and the C-terminal primer 5!-CGTACGCGGATCCTATTTAACGCGCGAGAAAA-3!. The PCR product was digested with NcoI and BamHI and ligated with the similarly restricted
plasmid pET 15b (Novagen). The ligated product was transformed in E.
coli DH5!. Clones were identified by PCR on the whole cells using the
T7 promoter primer 5!-TAATACGACTCACTATA-3! and the C-terminal
8835
8836
E. coli 23 S rRNA m5U1939 Methyltransferase
primer used above. Plasmids were isolated from the clones containing
the desired PCR product and confirmed by DNA sequencing performed
at the Biomolecular Resource Center, UCSF (San Francisco, CA). The
resultant construct was named pRJR. Molecular biology procedures
have been described elsewhere (22).
Expression and Purification—pRJR was transformed into E. coli
BL21(DE3) pLysS cells. Cells were grown at 37 °C in Luria Bertani
medium containing ampicillin (100 "g/ml) to an optical density of 0.6 at
600 nm. Isopropyl-1-thio-#-D-galactopyranoside was added to a final
concentration of 1 mM, and cells were grown for an additional 3 h before
harvesting. The cell pellet was suspended in buffer A (20 mM potassium
phosphate, pH 7.0, in 10% glycerol) containing 50 mM NaCl and the
protease inhibitor mixture (Complete-Mini™, Roche Molecular Biochemicals) was added. Cells were lysed by two passes through a French
press at 16,000 p.s.i., and debris was removed by centrifugation at
15,000 # g for 15 min. The supernatant was loaded on a SP-Sepharose
column, equilibrated with 100 mM NaCl in buffer A, and the column was
washed before eluting with a gradient of 100 –700 mM NaCl in buffer A.
Fractions containing RumA were identified by polyacrylamide gel electrophoresis and pooled. The pooled fractions were diluted 1:1 with
buffer A and loaded onto a hydroxyapatite column equilibrated with 50
mM potassium phosphate buffer, pH 7.0, in 10% glycerol. The column
was washed and then eluted with a gradient of 100 – 450 mM potassium
phosphate buffer, pH 7.0, in 10% glycerol. Fractions containing RumA
were pooled, and 3 M (NH4)2SO4 was added to the pool to bring the
(NH4)2SO4 concentration to 1 M. This pool was loaded onto a column of
phenyl-Sepharose equilibrated with 1 M (NH4)2SO4 in buffer A. The
column was eluted with a gradient of 0.6 – 0 M (NH4)2SO4 in buffer A.
Fractions were pooled and concentrated using a Centriprep concentrator (Amicon). The purified protein was analyzed by N-terminal sequencing for 10 cycles of Edman degradation at the Beckman Center, Stanford University.
RNA Synthesis—Unmodified RNA substrates were synthesized by
runoff transcription of appropriate plasmids using MEGAscript in vitro
transcription kit (Ambion, TX). p67YF0 (23) digested with BstNI, pWK1
(24) digested with AflII, and pCW1 (25) digested with Bsu 36I were used
as templates for the syntheses of tRNA, 16 S rRNA, and 23 S rRNA,
respectively. Ammonium acetate was added to a final concentration of
0.5 M, and the reaction mixture was extracted successively with phenol
and chloroform and precipitated with 1 volume of isopropyl alcohol.
Transcripts were dissolved in water and separated from any remaining
unincorporated nucleotides by passing through Nuc-away spin columns
(Ambion, TX). Chemically synthesized RNA oligonucleotides were purchased from Dharmacon Research Inc., and were deprotected using
their recommended procedure. After deprotection, RNA was dried using
a Speed Vac concentrator and dissolved in water before use.
Concentrations of tRNA, 16 S rRNA, and 23 S rRNA were calculated
using the reported A260 unit values of 1600, 67, and 40 pmol, respectively (26). Concentrations of synthetic RNA, 23 S fragment-(736 –760),
and 23 S fragment-(1930 –1969) were calculated using A260 unit value of
3860 and 2536 pmol, respectively, as provided by the manufacturer.
Measurement of the Extinction Coefficient—The extinction coefficient
of RumA at 280 nm was determined using the method of Gill and von
Hippel (27) assuming all the cysteines were in the thiol form. Absorption spectra were recorded on a HP8452A diode array spectrophotometer. The measured $280 value for the native protein was 41,500
$1
M
cm$1.
Methylation Assays—Methylation assays were done by minor modifications of the procedure of Gu et al. (18). Methylation reaction mixtures contained 1 "M RNA, 50 "M [3H-Me]AdoMet (1 Ci/0.2 mmol), 400
units/ml of RNasin, and 0.25 "M enzyme in reaction buffer (200 mM
Tris-HCl, pH 7.5, 80 mM NH4Cl, 4 mM magnesium acetate, and 12 mM
dithiothreitol). The reaction was performed at 37 °C and quenched at
the desired time points by adding 20 "l of aliquots to 0.5 ml of 100 mM
glycine-HCl buffer, pH 2.3. This mixture was adsorbed on DEAE papers, and the papers were washed with glycine-HCl buffer followed by
ethanol and counted for tritium.
Kinetic constants were measured by pseudo first order MichaelisMenten kinetics. To obtain the Km for RNA, AdoMet concentration was
kept at 150 "M, and initial velocities were measured at varying concentrations of RNA (23 S rRNA fragment-(1930 –1969)) from 0.5 to 14 "M.
To obtain the Km for AdoMet, RNA concentration was kept at 25 "M,
and initial velocities were measured at varying concentrations of
AdoMet from 2 to 160 "M. Enzyme concentration was 0.13 "M.
Nucleoside Analysis—In vitro transcribed 23 S rRNA was methylated using [3H-Me]AdoMet, extracted with phenol and precipitated
twice with ethanol. The [3H-Me]-methylated RNA was digested with
nuclease P1 followed by calf intestine alkaline phosphate. Unlabeled
3-methyluridine, 5-methyluridine, and 5-methylcytidine were added to
the digested RNA, and the mixture was separated on a Hewlett Packard
1090 HPLC system equipped with a reverse-phase C18 ion-pair column
(Beckman) using the following conditions: solvent A, 0.25 M NH4(OAC),
pH 6.0; solvent B, 40% CH3CN; gradient: 0 min, 0% B; 10 min, 5% B; 25
min, 15% B; 30 min, 50% B; flow rate: 1 ml/min. Column eluant was
monitored at 260 nm, 1-ml fractions were collected and counted in 5 ml
of scintillation mixture (26).
Nuclease Protection Assay—In vitro transcribed 23 S rRNA was
methylated using [3H-Me]AdoMet as described above. 12 pmol of methylated RNA and a 66-fold molar excess of DNA oligonucleotide complementary to the region 737–757 (5!-TAATTTTTCAACATTAGTCGG-3!)
or 1929 –1949 (5!-CCGACAAGGAATTTCGCTACC-3!) were added to
110 "l of hybridization buffer (40 mM MES, pH 6.0, 400 mM NaCl, 1 mM
EDTA, 80% (v/v) formamide), heated at 90 °C for 10!, annealed at 52 °C
overnight, and cooled slowly to room temperature. The hybridization
mixture was diluted 10-fold in nuclease reaction buffer (20 mM Tris-Cl,
pH 7.5, 150 mM NaCl, 5 mM EDTA) and digested with 200 units of
RNase T1 for 30 min at room temperature. The RNase-digested material was diluted 4-fold with 10 mM Tris-HCl, pH 7.5, adsorbed on DEAE
paper, washed with a solution of 10 mM Tris-Cl, pH 7.5, 200 mM NaCl,
and counted for adsorbed radioactivity. A control experiment with no
DNA oligonucleotide was also performed.
Site-directed Mutagenesis—Site-directed mutagenesis was performed on plasmid pCW1 encoding 23 S rRNA using the QuikChange
kit (Stratagene). Mutagenic primers for the U747C mutation were 5!GACCGAACCGACTAATGTCGAAAAATTAGCGGATGAC-3! and 5!GTCATCCGCTAATTTTTCGACATTAGTCGGTTCGGTC-3!, and
mutagenic primers for the U1939C mutation were 5!-GGTCCTAAGGTAGCGAAACTCCTTGTCGGGTAAGTTC-3! and 5!-GAACTTACCCGACAAGGAGTTTCGCTACCTTAGGACC-3!. The site of mutation
is underlined. The presence of the mutation was confirmed by DNA
sequencing.
Metal Analysis—Metal analysis was performed by inductively coupled plasma emission spectroscopy at the Garatt-Callahan Company
(Millbrae, CA). The protein sample was extensively dialyzed against 50
mM ammonium acetate buffer. The dialysis buffer was stirred for 2 h
with 10 g/liter Chelex-100 sodium form (Bio-Rad) to remove divalent
cations and filtered. All glassware was washed with 20% HNO3 and
rinsed with Chelex-treated water.
RESULTS
Cloning and Expression
The rumA (ygcA) ORF was cloned into pET 15b using PCR.
DNA sequence analysis revealed a single base mutation, nucleotide T78G. The mutation was at the wobble position and did
not affect the resulting amino acid sequence. This variation in
sequence could be because of a PCR error or polymorphism in
the population. Cells containing the clone expressed a 50-kDa
protein upon induction with isopropyl-1-thio-#-D-galactopyranoside. This 50-kDa protein band, which was visible on SDSPAGE, was absent in the lysate of the cells collected prior to the
induction (data not shown).
Purification and Identification
Protein was purified using successive chromatography steps
of SP-Sepharose, hydroxyapatite, and phenyl-Sepharose columns as described under “Experimental Procedures.” A yield of
approximately 2.5 mg of purified protein was obtained from one
liter of E. coli culture. The purity of the protein was estimated
by SDS-PAGE to be %95% (Fig. 1).
The purified protein was subjected to 10 cycles of Edman
degradation. The N-terminal sequencing of the protein yielded
the sequence AQFYSAKRRT. The experimentally obtained sequence matched the sequence in the data base (Swiss-Prot
accession number P55135) with the exception that the N-terminal initiator methionine was absent. The N-terminal methionine is often cleaved from mature proteins when the residue
following it has a small side chain (29) such as the alanine in
RumA.
E. coli 23 S rRNA m5U1939 Methyltransferase
FIG. 1. SDS-PAGE analysis of RumA. Lane 1, crude soluble lysate
of isopropyl-1-thio-#-D-galactopyranoside-induced BL21(DE3) plysS E.
coli containing pRJR; lane 2, pooled fractions from the SP-Sepharose
column; lane 3, pooled fractions from hydroxyapatite column; lane 4,
pooled fractions from phenyl-Sepharose column. 4 "g protein was
loaded onto each lane. Lane M, 10-kDa molecular mass ladder (Invitrogen). Position of the 50-kDa protein standard is indicated.
8837
FIG. 2. Methylation assay of unmodified RNA substrates. In
vitro transcribed E. coli 23 S rRNA (E), 16 S rRNA (!), and yeast
tRNAphe (") were methylated using [3H-Me]AdoMet. The extent of the
reaction was measured as incorporation of [3H] into RNA and was
monitored by a DEAE filter binding assay. The concentrations of RNA,
AdoMet, and enzyme were 1 "M, 50 "M, and 0.25 "M, respectively.
Identification of RNA Substrate
In vitro transcribed 23 S rRNA, 16 S rRNA, and yeast
tRNAPhe were assayed as possible substrates for catalysis by
RumA (Fig. 2). Only reactions with 23 S rRNA showed significant methylation activity. Approximately 0.8 mol of methyl
group was incorporated/mol 23 S rRNA. A value close to 1 mol
of methylation/mol 23 S rRNA was obtained when a high concentration of enzyme was used (data not shown). There was no
detectable methylation of tRNA, and &0.02 mol of methyl
group was incorporated/mol 16 S rRNA under the conditions
examined. These results indicate that 23 S rRNA is the preferred substrate for RumA, and most probably, it is modified at
only one position.
Product Identification by Nucleoside Analysis
23 S rRNA was methylated [3H-Me] using AdoMet and was
enzymatically hydrolyzed to constituent nucleosides. This nucleoside mixture along with exogenously added 3-methyluridine, 5-methyluridine, and 5-methylcytidine as standards was
analyzed on reverse-phase HPLC (Fig. 3). The m5U peak eluted
at approximately 17 min. More than 90% total radioactivity
co-eluted with the m5U peak. This result established that m5U
or ribothymidine is the product of the RumA-catalyzed
modification.
Position of Modification
E. coli 23 S rRNA contains two m5Us at positions 747 and
1939. The site modified by RumA was identified by nuclease
protection assay and by the reaction of the enzyme with the
site-specific mutants of the substrate RNA.
Nuclease Protection Assay—Methylated [3H-Me]23 S rRNA
was hybridized with 21-residue single-stranded DNA oligonucleotides complementary to either the U747 region or the
U1939 region and digested with RNase T1. The RNA protected
from digestion was monitored by a DEAE filter binding assay
(Fig. 4). When the oligonucleotide complementary to the sequence 1929 –1949 of 23 S rRNA was used for hybridization,
%85% radioactivity was protected and retained on the filter,
whereas only 25% radioactivity was retained when oligonucleotide complimentary to the region of U747 was employed for
hybridization. In a control experiment in which no oligonucleotide was used, '10% radioactivity was retained on the DEAE
FIG. 3. Identification of the nucleoside product. [3H-Me]-methylated 23 S rRNA was hydrolyzed to the constituent nucleosides by
using nuclease P1 and alkaline phosphatase. Methylated nucleosides
5-methylcytidine (m5C), 5-methyluridine (m5U), and 3-methyluridineas (m3U) were added after the reaction as standards, and the mixture
was separated on HPLC. 1-ml fractions were collected and counted for
3
H. The radioactivity profile was overlaid on the HPLC chromatogram.
————
, A260; . . . ! . . . , disintegration per minute (DPM).
filter. These results suggested that U1939 or its neighboring
residues is harboring the [3H-Me]methyl group.
Methylation of RNA Mutants—To verify the position of the
modified nucleotide, we constructed two single mutants of 23 S
rRNA in which either uridine 747 or uridine 1939 was mutated
to cytidine. The ability of these two mutants to serve as substrate for RumA was assayed (Fig. 5). In 23 S rRNA U747C,
0.75 mol of methyl group was incorporated/mol of RNA under
the conditions examined. There was no detectable methylation
of 23 S rRNA U1939C. These results confirmed that U1939 and
not U747 is the target for methylation by RumA.
Methylation of RNA Fragments
We obtained two synthetic fragments of E. coli 23 S rRNA
corresponding to sequence 736 –760 and 1930 –1969, respectively. These fragments were designed based on the structure
of the homologous regions of the 50 S subunit of ribosome from
Haloarcula marismortui (15) and were predicted to have an
independently folded structure. We tested these two fragments
for their ability to serve as substrates for RumA (Fig. 6). The
fragment corresponding to the 23 S rRNA sequence 736 –760
was not methylated by the enzyme. The RNA oligonucleotide
corresponding the 23 S rRNA sequence 1930 –1969 was a good
8838
E. coli 23 S rRNA m5U1939 Methyltransferase
FIG. 4. Nuclease protection assay. [3H-Me]-methylated 23 S rRNA
was hybridized with the DNA oligonucleotides complementary to the
regions of U747 or U1939. The hybridized RNA was digested with
RNase T1 and then adsorbed onto the DEAE filter. The radioactivity
retained on the filter was measured. Values shown are the average of
three measurements. Open bars, undigested; solid bars, digested with
RNase T1.
FIG. 5. Methylation of 23 S rRNA mutants. U747C 23 S rRNA (E)
and U1939C 23 S rRNA (!) was methylated using [3H-Me]AdoMet. The
extent of the reaction was measured as an incorporation of [3H] into
RNA and monitored by a DEAE filter binding assay. RNA, AdoMet, and
enzyme concentrations were 1, 50, and 0.25 "M, respectively.
substrate for the enzyme and could be fully modified at a rate
equivalent to that observed with full-length 23 S rRNA.
The apparent Km values for the 40-mer RNA oligonucleotide
and AdoMet were 3 and 26 "M, respectively, and the apparent
kcat was approximately 0.06 s$1.
Identification of the Bound Prosthetic Group
Purified preparations of RumA were brownish-yellow, indicating the presence of a bound prosthetic group that was identified empirically by the absorption spectrum and was further
confirmed by metal analysis employing inductively coupled
plasma emission spectroscopy.
Absorption Spectrum—The absorption spectrum of the enzyme (Fig. 7) showed the usual protein aromatic band at 280
nm. There was also a shoulder at around 325 nm, a broad
longer wavelength absorption band from approximately 350 to
400 nm, and a long tail extending well beyond 500 nm. This
spectrum is reminiscent of that reported for aconitase (30),
which contains an iron-sulfur cluster. The spectral similarity
between RumA and aconitase suggested the presence of bound
FIG. 6. Methylation of RNA oligonucleotides. 23 S rRNA fragments corresponding to sequence 736 –760 ("), 1930 –1969 (!), and
full-length 23 S rRNA (E) were methylated using [3H-Me]AdoMet. The
extent of the reaction was measured as an incorporation of [3H] into
RNA and monitored by a DEAE filter binding assay. RNA, AdoMet, and
enzyme concentrations were 1, 50, and 0.25 "M, respectively.
FIG. 7. Absorption spectrum of RumA. Absorption spectrum of the
protein was recorded in 20 mM potassium phosphate buffer, pH 7.0, 100
mM NaCl. Protein concentration was 21.6 "M.
iron in the enzyme. The extinction coefficient at 350 nm was
measured as 13,100 M$1 cm$1.
Metal Analysis—A RumA preparation was dialyzed against
divalent metal free ammonium acetate buffer and analyzed for
the presence of 19 different metal ions by inductively coupled
plasma emission spectroscopy. The results confirmed the presence of iron in the sample. The stoichiometry was calculated as
1.9 equivalents of iron/mol of enzyme based on the measured
$280 value of 41,500 M$1 cm$1. No other divalent cation was
detected in the sample. The only other metal observed was
sodium, which was a result of the treatment of the dialysis
buffer with the sodium form of Chelex 100. The dialysis buffer
analyzed as a control also contained sodium and no other metal
ion. Mass spectrometry ruled out the presence of any other
prosthetic groups such as pyridoxal phosphate, which might
have conferred the yellow color (data not shown).
DISCUSSION
This report demonstrates that RumA is an AdoMet-dependent methyltransferase whose substrate is 23 S rRNA. The
enzyme does not act upon tRNA or 16 S rRNA. Nucleoside
analysis showed the presence of m5U, indicating that RumA
methylates the C-5 of the uridine ring. The target nucleotide
E. coli 23 S rRNA m5U1939 Methyltransferase
for the enzyme was identified as U1939 by a RNase protection
assay and more conclusively by methylation assays on sitespecific mutants of 23 S rRNA. The enzyme is able to efficiently
utilize a 40-mer RNA fragment and does not require a complete
23 S rRNA nor any ribonucleoprotein particle for its activity. It
was suggested previously that the product of ygcA ORF (RumA)
was an AdoMet-dependent methyltransferase (31), and it was
further predicted that RumA was an m5U-methyltransferase
that would act on 23 S rRNA (21). The results in this study
confirm the predictions made by the bioinformatics-based approach. An intriguing finding of this work is the presence of two
bound iron atoms in each monomer of RumA. This is the first
report of a methyltransferase that binds iron.
The mechanism of the tRNA m5U54 methyltransferase,
RUMT, has been investigated in detail (19). The catalytic
mechanism of RUMT involves a nucleophilic attack at the C-6
of the uridine by the thiolate of Cys-324, direct SN2 transfer of
the methyl group from AdoMet to the C-5 of uridine followed by
the abstraction of the proton at C-5 and finally the #-elimination of the enzyme to yield m5U. Amino acid sequence comparison of RumA with RUMT shows that Cys-389 is the catalytic
nucleophile of RumA, and sequence similarity throughout the
catalytic domain strongly suggests that the two enzymes share
a common mechanism. This mechanism does not invoke involvement of metal ions in the reaction. Hence, it is unlikely
that iron is required for the methyltransferase reaction.
AdoMet-dependent methyltransferases have a modular architecture where a common catalytic core and a variable substrate recognition module unite to facilitate the catalysis of a
wide variety of substrates (32, 33). The alignment of RUMT,
RumA, and other putative m5U RNA methyltransferases
shows that the catalytic domain containing the AdoMet binding
motifs and the catalytic cysteine is in the C-terminal region of
these sequences (21). The N-terminal extension of RumA contains a sequence motif, C-5X-C-2X-C (residues 81–90), which
may be the iron-binding site. Similar motifs have been documented as the signature of the iron-sulfur binding region in
ferredoxins (Prosite accession number PDOC 00175) (34) and
other Fe-S proteins (35). This iron-binding motif is conserved in
sequences with high homology to RumA and other putative
rRNA m5U methyltransferases, but it is absent in RUMT and
other putative tRNA m5U methyltransferases.2 It is possible
that the iron-binding site forms part of the RNA recognition
domain.
Several RNA modifications and modification enzymes have
been well characterized, but the knowledge of their biological
role is mostly obscure. Certain tRNA modifications have been
reported to increase the fidelity and efficiency of translation
(36). In many cases, deletion mutants for these enzymes did not
show significant growth rate defects when grown in either
minimal or rich media. These mutants, however, had a selective disadvantage when grown in competition with the wildtype cells (7). trmA-encoding tRNA m5U54 methyltransferase
and rluD(yfiI)-encoding pseudouridine (") synthase for "1911,
1915, and 1917 have been found to be essential for the viability
and/or growth of cells (20, 37, 38). However, the deficiency in
these deletion strains could be resurrected by substitution with
genes encoding inactive mutants (20, 39). These reports indicated the possibility of yet unknown second function for some of
the RNA-modifying enzymes.
2
S. Agarwalla, unpublished observation. BLAST searches on Swiss
Protein and trembl databases were performed at the Swiss Institute of
Bioinformatics using BLAST network service on 12/06/01. RumA, E.
coli ORF ybjF, and RUMT sequences were used as probes. Nineteen
sequences from three searches with scores higher than 175 were
aligned using the software T-COFFEE (www.ch.embnet.org/
software/TCoffee.html).
8839
The physiological relevance of RumA and m5U1939 is unknown, however it is tempting to propose an association with
the RelA-dependent “stringent response.” This proposal is
based in part on the location of U1939 in the ribosome. In the
70 S ribosome structure, the conserved loop containing
U1939 tucks into the major groove at the end of the acceptor
stem of the A-site tRNA (16), and hence it is strategically
located for sensing the presence of uncharged tRNA at this
site in the ribosome. During amino acid starvation, the presence of uncharged tRNA at the A-site activates the ribosome
bound RelA, resulting in the increased levels of the nucleotide (p)ppGpp, the mediator of the stringent response (40).
Moreover, rumA and relA are contiguous genes on the E. coli
chromosome separated by &50 bases and transcribed in the
same direction (28). The identification of the methyltransferase gene for m5U1939 has paved the way for further studying the physiological role of this modification situated at an
important site of the ribosome.
Acknowledgments—We sincerely thank Patricia J. Greene for carefully reading and editing the manuscript and Maria Elena-Diaz for
technical assistance. We also thank the UCSF Mass Spectrometry
Facility for mass spectrometric analyses.
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