|
| |
Journal of Bacteriology, May 2003, p . 3238-3243, Vol . 185,
No . 10
The
yggH Gene of Escherichia coli Encodes a tRNA (m7G46)
Methyltransferase
Lara G . S . De Bie,1 Martine Roovers,1 Yamina
Oudjama,2 Ruddy Wattiez,3 Catherine Tricot,2
Victor Stalon,1,2 Louis Droogmans,1*
and Janusz M . Bujnicki4
Laboratoire de Microbiologie, Université Libre de Bruxelles,1
Institut de Recherches Microbiologiques Jean-Marie Wiame, B-1070 Brussels,2
Laboratoire de Chimie Biologique, Université de Mons-Hainaut, B-7000 Mons,
Belgium,3 Bioinformatics Laboratory, International Institute of
Molecular and Cell Biology, 02-109 Warsaw, Poland4
Received 20 September 2002/ Accepted 3 March 2003
We cloned, expressed, and purified the Escherichia coli YggH
protein and show that it catalyzes the S-adenosyl-L-methionine-dependent
formation of N7-methylguanosine at position 46 (m7G46)
in tRNA . Additionally, we generated an E . coli strain with a
disrupted yggH gene and show that the mutant strain lacks tRNA
(m7G46) methyltransferase activity .
About 30 different modified nucleosides have been identified in
Escherichia coli tRNA . Methylation is one of the most common
modifications, and several mutants affected in tRNA methylation have
been obtained (5) . However, only a few E . coli tRNA
methyltransferase (MTase) genes have been cloned and characterized:
trmA, trmD, and trmH are involved in the
formations of m5U54, m1G37, and Gm18,
respectively (8, 15, 16) .
Several tRNA MTases have been purified, and the corresponding genes
have been mapped on the E . coli chromosome (5,
13), but it has not been convincingly shown which
open reading frame (ORF) encodes a given enzyme . On the other hand,
evolutionary relationships among various RNA MTase families have been
studied and predictions of novel specificities for uncharacterized
ORFs have been made (3) . Nevertheless, there are
still missing links between many known enzymatic activities and
predicted RNA MTase genes .
As part of a large-scale project aimed at the identification and
classification of novel RNA MTases among the uncharacterized or
putative proteins in sequence databases, we analyzed the product of
the E . coli yggH ORF . This protein exhibits similarity to S-adenosyl-L-methionine
(AdoMet)-dependent MTases in the predicted cofactor-binding region
but shares no specific amino acid signatures with other families of
RNA MTases in the predicted catalytic region, suggesting that it may
encode an RNA MTase with a novel specificity . Thus, we selected it
for experimental characterization .
Amplification and cloning of the yggH ORF. The
yggH ORF was PCR amplified from E . coli genomic DNA (strain
XL1-Blue) by using Pfu DNA polymerase (Promega) . The primers
(Table 1) were designed to amplify the yggH ORF
with its ribosome binding site . Primers LDB1 and LDB3 were used for
the production of a recombinant YggH protein bearing a C-terminal His
tag (YggHH6) . Primers LDB1 and LDB2 were used for the production of
the untagged YggH .
| TABLE 1 . Oligonucleotides
|
|
The PCR products were cloned into the pCR-BluntII-TOPO vector
(Invitrogen) according to the manufacturer's instructions, generating
the pCR-yggHH6 and pCR-yggH plasmids (the strains and plasmids
used are shown in Table 2) . The XbaI/XhoI
insert of the pCR-yggHH6 plasmid was subcloned into the
corresponding sites of the pET30b overexpression vector (Novagen),
generating the pET-yggHH6 plasmid .
| TABLE 2 . Bacterial strains and plasmids
|
|
Expression and purification of the YggHH6 recombinant protein.
The YggHH6 protein was expressed in E . coli strain BL21(DE3) .
Transformed cells were grown at 37°C in Luria broth (supplemented
with kanamycin at 30 µg/ml) to an optical density at 660 nm of 0.7 .
At this stage, IPTG (isopropyl-ß-D-thiogalactopyranoside)
was added to a final concentration of 0.5 mM to induce recombinant
protein expression . Cells were harvested after 3 h of incubation
at 37°C, resuspended in buffer A (50 mM Tris-HCl [pH 7.5], 10
mM MgCl2, 10% glycerol), and lysed by sonication . The lysate
was cleared by centrifugation (20,000 x
g for 10 min) and was applied to a column of Chelating
Sepharose Fast Flow (Pharmacia Biotech) charged with Ni2+ .
The column was washed with buffer A supplemented with 5 mM imidazole,
and the adsorbed material was eluted with a linear gradient (0.05 M
up to 0.4 M) of imidazole . Eluted fractions were analyzed by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . At
this stage, the YggHH6 preparation contained several minor
contaminants (data not shown) .
YggHH6 was further purified by gel filtration chromatography . The
partially purified enzyme was dialyzed against buffer A supplemented
with 200 mM imidazole to keep the protein soluble and was applied on
a Superdex 200 column (Pharmacia Biotech) equilibrated with the same
buffer . SDS-PAGE analysis of the fractions containing YggHH6 showed
two discrete bands (Fig . 1A), both of which
corresponded to the YggH protein as demonstrated by mass spectrometry
fingerprint analysis . A similar mass fingerprint was obtained for
both bands, except for the C-terminal tag tryptic peptide, which was
absent for the lower band (result not shown) . Thus, the lower band
most probably corresponds to a degradation product of YggHH6, lacking
the C-terminal His tag . Gel filtration chromatography revealed that
the apparent molecular mass of the YggHH6 protein is about 27 kDa .
This shows that the protein exists as a monomer .
|
FIG . 1 . The product of the E . coli yggH ORF catalyzes the
formation of m7G in tRNA . (A) SDS-PAGE of the purified YggHH6
protein . Lane 1, molecular mass markers in kilodaltons (Pharmacia
Biotech); lane 2, purified protein . The thick and thin arrows indicate
the recombinant YggHH6 protein and its minor contaminant, respectively
(see the text for details) . (B) Autoradiography of a two-dimensional
chromatogram of 5' phosphate nucleotides on a thin-layer cellulose
plate . Total tRNA (100 µg) from the methionine-starved P4X-SB25 strain
was incubated in a 200-µl reaction mixture containing 50 mM PIPES
[piperazine-N,N'-bis(2-ethanesulfonic acid)]-Na (pH 7.0),
4 mM MgCl2, 10 µM [methyl-14C]AdoMet (53
mCi/mmol), and 0.4 µg of the purified YggHH6 protein . After a 30-min
incubation at 37°C, the tRNA was recovered and digested by nuclease P1,
and the resulting nucleotides were analyzed as described previously (12).
|
|
yggH encodes an MTase responsible for the formation of m7G46
in the variable loop of tRNA. To determine whether the product of the
yggH ORF was an MTase acting on tRNA, the purified YggHH6
protein was incubated with 14C-radiolabeled AdoMet (S-adenosyl-L-[methyl-14C]methionine)
and total tRNA was extracted from a methionine-starved P4X-SB25
strain (an E . coli met mutant relA strain) . After incubation,
the tRNA was hydrolyzed by nuclease P1 and the resulting nucleotides
were analyzed by bidimensional cellulose thin-layer chromatography
(2D-TLC) followed by autoradiography . The result shown in Fig .
1B revealed the formation of a single radioactive compound
with migration characteristics similar to those of 7-methylguanosine
5'-phosphate (pm7G) . Examination of the tRNA sequence database
(http://www.uni-bayreuth.de/departments/biochemie/trna/)
revealed that m7G is found only at position 46 (in the
variable loop) in 23 E . coli tRNA species, including
 (Fig.
2A) . Therefore, an in vitro transcribed
was
tested as a substrate for the purified YggHH6 protein . The metT
gene, encoding
,
was PCR amplified from E . coli genomic DNA by using primers
LDB4 and LDB5 . These primers were designed to amplify a 150-bp
fragment containing the metT gene . A second PCR was performed
on this 150-bp fragment by using primers LDB6 and LDB7 . The second
couple of primers was designed to introduce a T7 RNA
polymerase-dependent promoter at the 5' end of the metT gene
and an MvaI restriction site at the 3' end . The use of two
consecutive PCRs was necessary, since on the E . coli
chromosome, the metT gene is in tandem with the metU
gene that also encodes
.
The final PCR product was cloned into the SmaI site of the pUC18
vector, generating the pYL6 plasmid . Transcripts of
were
generated by using T7 RNA polymerase and MvaI-digested pYL6 as
the template as described previously (17) . Full-length
transcripts were purified by 10% PAGE .
|
FIG . 2 . In vitro-transcribed E . coli
is
a substrate of the YggH MTase . (A) Cloverleaf representation of the
nucleotide sequence of E . coli
(9) .
(B) Autoradiography of two-dimensional chromatograms of 5' and 3'
phosphate nucleotides on thin-layer cellulose plates . [ -32P]GTP-labeled
(a and d) or [-32P]UTP-labeled
(b and e) in vitro-transcribed
and
[-32P]GTP-labeled
in vitro-transcribed
 (c
and f) (106 cpm) were incubated in the presence (d, e, and f)
or absence (a, b, and c) of the YggHH6 protein . The reaction mixture
contained 50 mM PIPES-Na (pH 7.0), 4 mM MgCl2, 50 µM AdoMet,
and 0.4 µg of the purified YggHH6 protein . After 30 min of incubation at
37°C, the tRNA was recovered and digested by nuclease P1 (a, c, d, and
f) or RNase T2 (b and e), and the resulting nucleotides were analyzed as
described in the legend to Fig . 1.
|
|
The purified YggHH6 protein was incubated with AdoMet and [-32P]GTP-labeled
in vitro-transcribed
.
After incubation, the tRNA was hydrolyzed using nuclease P1, and the resulting
5' phosphate nucleotides were analyzed by 2D-TLC and autoradiography .
The results showed the formation of m7G in the incubated tRNA
(Fig . 2B) . To further confirm that m7G
formation occurs at position 46, a similar experiment was performed
using [-32P]UTP-labeled
.
After incubation in the presence of AdoMet and purified YggHH6, the tRNA was
hydrolyzed by RNase T2 . The analysis of the resulting 3' phosphate
nucleotides revealed the formation of m7G in the tRNA
(Fig . 2B), demonstrating that the m7G
produced by YggHH6 is 5' adjacent to a uridine . In the
,
several guanosines are 5' adjacent to a uridine . To further confirm that m7G
formation occurs at position 46, a mutant
in
which G46 was mutated into A
 ]
was generated by site-directed mutagenesis on the pYL6 plasmid by using primers
LDB8 and LDB9 . Transcripts of tRNAmMet(G46A) were obtained as
described above . The purified YggHH6 protein was incubated with
AdoMet and [-32P]GTP-labeled
in vitro-transcribed
.
After incubation, the mutant tRNA was hydrolyzed using nuclease P1, and the
resulting 5' phosphate nucleotides were analyzed by 2D-TLC and
autoradiography . The results showed that m7G was not found
in the incubated mutant tRNA (Fig . 2B) . Altogether,
these results strongly suggest that the purified YggHH6 recombinant
protein catalyzes the formation of m7G at position 46 in
E . coli tRNA .
The E . coli yggH gene is not essential for growth.
The E . coli yggH gene was inactivated by the insertion of an
ampicillin resistance (Apr) cassette . This was achieved by homologous
recombination, depending on bacteriophage
recombination functions present in the host strain (18) .
A linear DNA fragment in which the ß-lactamase gene is flanked by 40
bp corresponding to the 5' and 3' ends of the yggH gene was
obtained by PCR using the oligonucleotides LDB10 and LDB11 as primers
and plasmid pUC18 as the template . The PCR product was used to
transform the DY330 F'(pro-lac) strain, and transformants were
selected for ampicillin resistance . The presence of the Apr
cassette in the yggH gene in the resulting RDB1 strain was
checked by PCR using oligonucleotides LDB12, LDB13, and LDB14 as
primers (result not shown) . To determine whether m7G46
formation was affected in the RDB1 strain, crude extracts of the
DY330 F'(pro-lac) and RDB1 strains were incubated with AdoMet
and [-32P]GTP-labeled
in vitro-transcribed
.
After incubation, tRNA was hydrolyzed by nuclease P1 and the nucleotides were
analyzed by 2D-TLC and autoradiography . The results shown in
Fig . 3 revealed the absence of m7G formation in
RDB1 extract . Moreover, when the RDB1 strain was transformed with
plasmid pCR-yggH, an extract of the resulting strain allowed m7G
formation (Fig . 3) . Also, total (crude) tRNA
extracted from the wild-type strain DY330 F'(pro-lac) was not
a substrate for the purified YggH enzyme, while tRNA from the RDB1
strain was an excellent substrate for this enzyme (data not shown) .
All these data further confirm the role of the YggH protein in the
formation of m7G in tRNA and show that the yggH
gene is not essential for growth .
|
FIG . 3 . The E . coli RDB1 strain with an inactivated yggH
gene lacks tRNA (m7G46) MTase activity . The panels show
autoradiography of two-dimensional chromatograms of 5' phosphate
nucleotides on thin-layer cellulose plates . [-32P]GTP-labeled
in vitro-transcribed
(106
cpm) was incubated with a crude extract of the DY330 F' strain (wild
type) (a), of the RDB1 strain (b), or of the RDB1/pCR-yggH strain
(c) . The reaction mixture contained 50 mM PIPES-Na (pH 7.0), 4 mM MgCl2,
50 µM AdoMet, and 100 µg of total protein . After 30 min of incubation at
37°C, the tRNA was recovered and digested by nuclease P1, and the
resulting nucleotides were analyzed as described in the legend to Fig.
1.
|
|
Interestingly, a trmB mutant (strain GM18) affected in the formation
of m7G in tRNAs was obtained in the 1970s (14) .
Surprisingly, the trmB mutation has been mapped at 6 min and
the yggH ORF maps at 66 min on the E . coli chromosome .
Because of this discrepancy, yggH cannot yet be renamed
trmB . A possible explanation for the absence of tRNA (m7G46)
MTase activity in the GM18 strain would be that trmB encodes a
factor influencing yggH expression . Alternatively, two tRNA (m7G46)
MTases might exist in E . coli, as has been suggested
previously (4) . However, the fact that the
inactivation of the yggH gene leads to a complete absence of
tRNA (m7G46) MTase activity does not support this hypothesis .
Further work is required to better characterize the trmB mutation .
Sequence analysis of the YggH MTase reveals a distinct family of m7G
MTases. Searches of the sequence database by using PSI-BLAST (2)
revealed that orthologs of the yggH gene are present in all
completely sequenced bacterial genomes and in crown eukaryotes
(animals, plants, and fungi), while they are absent from all archaea
(data not shown; see also the National Center for Biotechnology
Information's COG database at
http://www.ncbi.nlm.nih.gov/cgi-bin/COG/palox?COG0220) . This
pattern of phylogenetic distribution is perfectly consistent with the
observed presence or absence of m7G in tRNAs from these
organisms (11) . Analysis of the multiple sequence alignment
(http://www.ncbi.nlm.nih.gov/COG/aln/COG0220.aln)
revealed typical MTase motifs in the YggH family and allowed
superimposition with the sequences of other m7G MTases
acting on different RNAs: the Agr family specific for G1405 within
bacterial 16S rRNA (7) and the Abd1 family specific
for the cap structure in mRNA (6) . The alignment of
representative members of the three m7G MTase families
(Fig . 4) revealed no striking similarities apart
from the residues important for the stability of the common fold or
forming the common cofactor-binding pocket . In particular, a
tetrapeptide in motif IV, which typically harbors catalytic residues
and is very similar in related MTases (10), exhibits
completely different patterns of conservation in YggH, Abd1,
and Agr, namely, PDPW, CLHY, and PCLE, respectively . It has been
argued that the Agr and Abd1 families may use different mechanisms of
guanine-N7 methylation, because the predicted
substrate-binding regions and catalytic sites of these enzymes are
dissimilar, even though they share a common structural core (7) .
Identification of the tRNA (m7G46) MTase activity of the
yggH ORF suggests a third, considerably diverged class of enzymes
that generate a similar product (m7G) within a distinct
macromolecular context . It remains to be determined whether these
three classes of enzymes exhibit any similarities in the m7G
methylation mechanism other than the use of a common cofactor and
whether they evolved from a common ancestor or independently from
various lineages of the MTase superfamily .
|
FIG . 4 . Sequence alignment of the representative members of three m7G
MTase families specific for tRNA, mRNA, and 16S rRNA: E . coli
YggH, S . cerevisiae Abd1p (cap 0 MTase family), and
Streptomyces kanamyceticus Kmr (Agr family) . Conserved motifs are
labeled according to the nomenclature used by Fauman et al . (10) .
The number of residues omitted for clarity is indicated in parentheses .
Conserved AdoMet-binding carboxylate residues are indicated by
asterisks, and conserved residues important for the stability of the
MTase fold are indicated with pluses.
|
|
ADDENDUM IN PROOF During the time in which this work was under
review, the tRNA (m7G46) MTase from the yeast
Saccharomyces cerevisiae was identified (1) .
Two proteins (Trm8p and Trm82p) forming a complex are required for m7G46
formation in yeast tRNA . Trm8p appears to be the yeast ortholog of
the E . coli YggH protein .
We thank D . Bregeon (Faculté de Médecine Necker Enfants Malades,
Paris, France) for the gift of the DY330 F'(pro-lac) strain,
R . Lavallée (Institut de Recherches Microbiologiques J.-M . Wiame) for
the SB25 strain, and G . Doumont (Université Libre de Bruxelles) for
constructing the pYL6 plasmid .
L.G.S.D.B . is a fellow of the F.R.I.A . (Fonds pour la Formation à
la Recherche dans l'Industrie et dans l'Agriculture), and L.D . and
R.W . are research associates of the F.N.R.S . (Fonds National de la
Recherche Scientifique) . J.M.B . is an EMBO/HHMI Young Investigator .
This work was supported by grants from the F.R.F.C . (Fonds pour la
Recherche Fondamentale Collective), the French Community of Belgium
(Actions de Recherches Concertées), and the Université Libre de
Bruxelles (Fonds E . Defay) .
* Corresponding author . Mailing address: Laboratoire de
Microbiologie, Université Libre de Bruxelles, Institut de Recherche
Microbiologiques J.-M . Wiame, 1, avenue E . Gryson, B-1070 Brussels, Belgium .
Phone: 32 2 526 7254 . Fax: 32 2 526 7273 . E-mail: louisd@dbm.ulb.ac.be .
- Alexandrov, A., M . R . Martzen, and E . M . Phizicky. 2002 .
Two proteins that form a complex are required for 7-methylguanosine
modification of yeast tRNA . RNA 8:1253-1266 .
- Altschul, S . F., T . L . Madden, A . A . Schaffer, J . Zhang, Z .
Zhang, W . Miller, and D . J . Lipman. 1997 . Gapped BLAST and PSI-BLAST: a
new generation of protein database search programs . Nucleic Acids Res . 25:3389-3402 .
- Anantharaman, V., E . V . Koonin, and L . Aravind. 2002 .
Comparative genomics and evolution of proteins involved in RNA metabolism .
Nucleic Acids Res . 30:1427-1464 .
- Aschhoff, H . J., H . Elten, H . H . Arnold, G . Mahal, W .
Kersten, and H . Kersten. 1976 . 7-Methylguanine specific
tRNA-methyltransferase from Escherichia coli . Nucleic Acids Res . 3:3109-3122.
- Björk, G . R. 1995 . Biosynthesis and function of modified
nucleosides in tRNA, p . 165-205 . In D . Söll and U . L . Rajbhandary
(ed.), tRNA: structure, biosynthesis, and function . ASM Press, Washington,
D.C.
- Bujnicki, J . M., M . Feder, M . Radlinska, and L . Rychlewski.
2001 . mRNA:guanine-N7 cap methyltransferases: identification of
novel members of the family, evolutionary analysis, homology modeling, and
analysis of sequence-structure-function relationships . BMC Bioinformatics
2:2.
- Bujnicki, J . M., and L . Rychlewski. 2001 . Sequence
analysis and structure prediction of aminoglycoside-resistance 16S rRNA:m7G
methyltransferases . Acta Microbiol . Pol . 50:7-17.
- Byström, A . S., and G . R . Björk. 1982 . Chromosomal
location and cloning of the gene (trmD) responsible for the synthesis
of tRNA(m1G) methyltransferase in Escherichia coli K12 . Mol .
Gen . Genet . 188:440-446.
- Cory, S., and K . A . Marcker. 1970 . The nucleotide
sequence of methionine transfer RNA-M . Eur . J . Biochem . 12:177-194.
- Fauman, E . B., R . M . Blumenthal, and X . Cheng. 1999 .
Structure and evolution of AdoMet-dependent MTases, p . 1-38 . In X .
Cheng and R . M . Blumenthal (ed.), S-Adenosylmethionine-dependent
methyltransferases: structure and functions . World Scientific Inc., Singapore.
- Grosjean, H., M . Sprinzl, and S . Steinberg. 1995 .
Posttranscriptionally modified nucleosides in transfer RNA: their locations
and frequencies . Biochimie 77:139-141.
- Keith, G. 1995 . Mobilities of modified ribonucleotides
on two-dimensional cellulose thin layer chromatography . Biochimie 77:142-144.
- Leung, H.-C . E., T . G . Hagervall, G . R . Björk, and M . E .
Winkler. 1998 . Genetic locations and database accession numbers of
RNA-modifying and -editing enzymes, p . 561-567 . In H . Grosjean and R .
Benne (ed.), Modification and editing of RNA . ASM Press, Washington, D.C.
- Marinus, M . G., N . R . Morris, D . Söll, and T . C . Kwong.
1975 . Isolation and partial characterization of three Escherichia coli
mutants with altered transfer ribonucleic acid methylases . J . Bacteriol .
122:257-265.
- Ny, T., P . H . R . Lindström, T . G . Hagervall, and G . R .
Björk. 1988 . Purification of transfer RNA (m5U54)-methyltransferase
from Escherichia coli . Association with RNA . Eur . J . Biochem . 177:467-475.
- Persson, B . C., G . Jager, and C . Gustafsson. 1997 . The
spoU gene of Escherichia coli, the fourth gene of the spoT
operon, is essential for tRNA (Gm18) 2'-O-methyltransferase activity .
Nucleic Acids Res . 25:4093-4097 .
- Sampson, J . R., and O . C . Uhlenbeck. 1988 . Biochemical
and physical characterization of an unmodified yeast phenylalanine transfer
RNA transcribed in vitro . Proc . Natl . Acad . Sci . USA 85:1033-1037.
- Yu, D., H . M . Ellis, E.-C . Lee, N . A . Jenkins, N . G .
Copeland, and D . L . Court. 2000 . An efficient recombination system for
chromosome engineering in Escherichia coli . Proc . Natl . Acad . Sci . USA
97:5978-5983 .
Free Online Full-text Article
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
|
