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Journal of Bacteriology, December 2001, p. 7397-7402, Vol. 183, No. 24
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.24.7397-7402.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
The Mere Lack of rT Modification in Initiator tRNA Does Not
Facilitate Formylation-Independent Initiation in
Escherichia coli
Swapna
Thanedar,
T. K.
Dineshkumar, and
Umesh
Varshney*
Department of Microbiology and Cell Biology,
Indian Institute of Science, Bangalore 560 012, India
Received 2 July 2001/Accepted 18 September 2001
 |
ABSTRACT |
Formylation of initiator methionyl-tRNA is essential for normal
growth of eubacteria. However, under special conditions, it has been
possible to initiate protein synthesis with unformylated initiator tRNA
even in eubacteria. Earlier studies suggested that the lack of
ribothymidine (rT) modification in initiator tRNA may facilitate
initiation in the absence of formylation. In this report we show, by
using trmA strains of Escherichia coli
(defective for rT modification) and a sensitive in vivo initiation
assay system, that the lack of rT modification in the initiators is not
sufficient to effect formylation-independent initiation of protein synthesis.
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TEXT |
In stark contrast to its
unformylated counterparts in archaea and eukarya, initiation of protein
synthesis in eubacteria, mitochondria, and chloroplasts occurs with
formylmethionyl (fMet)-tRNA (10). Therefore, this distinct
mechanism of initiation with fMet-tRNA in eubacteria has been of
interest for over three decades (1, 3, 5, 8, 12, 14, 18, 22, 23,
24, 25, 26). Formylation of the initiator tRNAs in eubacteria
utilizes formyltetrahydrofolate (fTHF) as a cofactor, and this
modification has been shown to be crucial in initiation of protein
synthesis (10, 15). Nevertheless, under special
conditions, it has been possible to initiate protein synthesis with
unformylated initiator tRNA even in eubacteria. For instance, with the
use of inhibitors of the folate biosynthesis pathway such as
trimethoprim or aminopterin it was possible to deplete
Streptococcus faecalis and Bacillus subtilis of
fTHF. Growth under these conditions required supplementation of the
medium with the end products of the other metabolic pathways that also
utilize fTHF as a cofactor. Interestingly, these bacteria grew
essentially normally even in the presence of the inhibitors (2,
18). Modified base analysis of the initiator tRNA from folate-deficient bacteria showed the lack of ribothymidine (rT) modification at position 54 in the highly conserved T
C sequence (5). This observation led to a suggestion that the lack of rT modification could somehow lead to formylation-independent initiation in gram-positive bacteria (18). Subsequently,
it was shown that at least in B. subtilis, the
U54-methyltransferase uses fTHF as a cofactor
(6, 7, 16). Thus, the lack of rT in tRNA could simply be a
consequence of depletion of fTHF in these bacteria when grown in the
presence of trimethoprim or aminopterin. Studies along these lines in
Escherichia coli resulted in the isolation of a mutant,
which was partially deficient in rT modification, and reciprocated with
poor growth in the presence of the inhibitors of fTHF biosynthesis
(3). Since in E. coli
S-adenosylmethionine (rather than fTHF) is used as a
cofactor by the U54-methyltransferase
(19), the lack of rT modification could be implicated in
formylation-independent initiation. This interpretation is consistent
with the observation that in the eukaryotic initiators, which are
naturally used in their unformylated state, position 54 is not a U, and
consequently these tRNAs lack rT at this position (22).
To study the role of the absence and/or deficiency of rT modification
in the initiator tRNAs in formylation-independent initiation in
eubacteria, we exploited the trmA strains of E. coli (defective for rT modification) by two approaches. First, we
analyzed the growth phenotypes of these strains in the presence of the
inhibitors of fTHF biosynthesis. In the second approach these strains
were used to analyze initiation activities of the formylation-defective initiator tRNAs in a highly sensitive initiation assay system (27). The latter approach allowed us to circumvent the use
of folate biosynthesis inhibitors in discerning the importance of the
lack of the rT modification in initiator tRNA in initiation.
Effect of folate biosynthesis inhibitors on growth of E.
coli trmA strains.
Two of the trmA strains,
KL356 (trmA14) and G11-5-18 (trmA5), defective in
U54 methyltransferase (4, 13) were
obtained from E. coli Genetic Stock Center and grown in
Luria-Bertani (LB) medium (supplemented with Gly, Ser, Met,
adenosine, thymidine, and pantothenate [3]) in the
absence or presence of various concentrations of the folate biosynthesis inhibitors (sulfathiazole and trimethoprim). As seen in
Fig. 1, the wild-type
(trmA+) or the trmA strains
grew equally well in the absence of the drugs (panels a to c).
Expectedly, the strain KL16 (trmA+) grew
very slowly in the presence of the inhibitors (panel a). In contrast to
the general expectation, even the trmA strains (KL356 and
G11-5-18) grew very slowly in the presence of the folate biosynthesis
inhibitors. In fact, the trmA strains were just as sensitive
to the drugs as the wild-type strain, suggesting that the lack of rT
modification in tRNA did not confer any growth advantage against the
inhibitors. The minimal growth of all of the strains (to similar
extent) at all concentrations of inhibitors suggests that it was most
likely due to the pools of fTHF present in the cells prior to the
addition of the inhibitors (Fig. 1a to c).
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FIG. 1.
Growth of E. coli KL16
(trmA+), KL356 (trmA14), and
G11-5-18 (trmA5) strains (a, b, and c,
respectively) in LB medium (17) supplemented with Gly (45 mg/liter), Met (50 mg/liter), Ser (50 mg/liter), adenosine (28 mg/liter), thymidine (4 mg/liter), and calcium pantothenate (0.4 mg/liter), in the absence or presence of the folate biosynthesis
inhibitors as shown (3). Amounts of trimethoprim and
sulfathiazole are per liter.
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Use of in vivo initiation assay to study initiation in E.
coli (trmA) with formylation-defective initiator
tRNAs.
The initiator tRNA mutant carrying a CUA anticodon change
(U35A36 mutations) initiates protein synthesis from UAG as an
initiation codon of the chloramphenicol acetyltransferase (CAT)
reporter and confers chloramphenicol resistance
(Cmr) to the host (27). Introduction
of the G72, or G72G73 mutations, into the U35A36 background renders
these tRNAs (G72/U35A36 and G72G73/U35A36 [Fig. 2, left panel])
inactive in initiation because of their defect at the step of
formylation (11). While the G72/U35A36 tRNA is more than
475-fold worse than the U35A36 tRNA in formylation, the G72G73 tRNA is
still worse, and its formylation cannot be detected in vitro. In
addition, the G72 substitution creates a 1:72 bp, which may make
the tRNA a substrate for peptidyl-tRNA hydrolase (9).
Thus, in vivo, there is no accumulation of the formylated forms of
these tRNAs (23, 26).
Both of the
trmA E. coli strains KL356 (
trmA14)
and G11-5-18 (
trmA5), defective in
U
54-methyltransferase, and the KL16 strain
(
trmA+) were transformed with the
constructs harboring the formylation-defective
tRNA mutants on the
plasmids pCATam1
metYCUA(G72) or
pCATam1
metYCUA(G72G73).
As these tRNAs
are aminoacylated by Gln, glutamine tRNA-synthetase
(GlnRS) was
overexpressed from a compatible plasmid (pACQS,
Kan
r) to help ensure their efficient
aminoacylation (Fig.
2,
right
panel [
27]). The initiation
activity of these tRNAs was examined
by their growth on chloramphenicol
plates and by the detection
of the reporter CAT protein in the cell
extracts by enzyme assays
and immunoblotting.
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FIG. 2.
Cloverleaf structure of the E. coli
initiator tRNA2fMet, indicating the sites of
mutations (left panel), and the binary plasmid system used for the in
vivo assay system (right panel). Plasmid
pCATam1metYCUA harbors the tRNAfMet
(U35A36, G72/U35A36, or G72G73/U35A36) and the reporter CATam1 genes,
whereas plasmid pACQS harbors the E. coli
glutaminyl-tRNA synthetase gene.
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|
Expectedly, all of the three strains of
E. coli, namely,
KL16 (
trmA+), KL356 (
trmA), and
G11-5-18 (
trmA) harboring the formylation-proficient
tRNA
(U35A36) on the CATam1 reporter plasmid (ampicillin resistant
[Amp
r]) showed growth on plates containing
ampicillin and kanamycin,
as well as on plates containing ampicillin,
kanamycin, and chloramphenicol
(Fig.
3,
sectors 1, 4, and 7). On the other hand, the transformants
harboring
the formylation-defective tRNAs (G72/U35A36 and G72G73/U35A36)
grew on
the plates containing ampicillin and kanamycin but failed
to grow on
the plates containing ampicillin, kanamycin, and chloramphenicol
(sectors 2, 5, and 8 and sectors 3, 6, and 9, respectively). Consistent
with the growth phenotype on the chloramphenicol plates, the
transformants
harboring the formylation-defective tRNAs showed only the
background
level (~0.07 to 0.15%) CAT activity with respect to the
U35A36
tRNA (Table
1) irrespective of the
nature of the
trmA allele.
To ascertain further that the
formylation-defective initiator
tRNAs failed to initiate in the
trmA strains of
E. coli, we performed
immunoblot
analysis (Fig.
4). In agreement with the
phenotypic
and the CAT assays, the band corresponding to CAT was seen
only
in the transformants containing the formylation-proficient
initiator
(lanes 1 and 4) and not in the ones harboring
formylation-defective
tRNAs (lanes 2 and 3 and lanes 5 and 6). However,
a band corresponding
to
-lactamase (Amp
r) was
seen in all of the lanes confirming the presence of
pCATam1
metYCUA (G72; G72G73) plasmids.
Importantly, this analysis ruled out overproduction
of CAT in inactive
form. Furthermore, the analysis of the total
tRNA from these
transformants by using acid urea gel Northern
blots (
26)
showed that the mature tRNA corresponding to the
G72/U35A36, as well as
the G72G73/U35A36, tRNA was produced and
aminoacylated in the cell
(Fig.
5). As expected, the U35A36 tRNA
was present predominantly in the formylated form (lanes 1 and
4), and
the G72/U35A36 and G72G73/U35A36 tRNAs were detected in
aminoacylated
and uncharged forms (lanes 2 and 5 and lanes 3 and
6, respectively).
Our other studies showed that the CATam1 reporter
gene in the
G72/U35A36 or the G72G73/U35A36 tRNA encoding plasmids
retains an
intact reading frame with UAG as an initiation codon
(
24,
25). Furthermore, as the initiation factor 2 (IF2) or
its
equivalents in eukaryotes and archaea are important in the
utilization
of initiator tRNAs in initiation, in yet another control
experiment, we
measured initiation activity of the G72G73/U35A36
tRNA in these strains
by CAT assays in the presence of IF2 overproduction.
While there was a
marginal increase in the initiation activity
of this tRNA in all of the
three strains, its activity in the
trmA strains (KL356 and
G11-5-18) did not increase beyond that
of the
trmA+ control strain (KL16) (data not
shown).
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FIG. 3.
E. coli transformants were streaked onto
2YT agar plates containing ampicillin and kanamycin (Kan + Amp) or
ampicillin, kanamycin, and chloramphenicol (Kan + Amp + Cm) and
incubated at 37°C for 18 h. The transformants of the strains
KL16 (sectors 1 to 3), KL356 (sectors 4 to 6), and G11-5-18 (sectors 7 to 9) contained the following plasmids: sectors 1, 4, and 7, pCATam1metYCUA and pACQS; sectors 2, 5, and 8, pCATam1metYCUA (G72) and pACQS; and sectors 3, 6, and 9, pCATam1metYCUA (G72G73) and pACQS.
Antibiotic concentrations were as follows: ampicillin, 100 µg/ml;
kanamycin, 25 µg/ml; and chloramphenicol, 30 µg/ml.
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TABLE 1.
Relative CAT activities in cell extracts of various
transformants containing formylation-proficient (U35A36) or-defective
(G72/U35A36 and G72G73/U35A36) tRNAs, along with the reporter CATam1
gene, in the strains of E. coli wild type (KL16) or mutants
containing different trmA alleles (KL356 and
G11-5-18)a
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FIG. 4.
The cell extracts (10 µg of total protein) prepared
from log-phase cultures were fractionated on a sodium dodecyl
sulfate-polyacrylamide gel (12%) and electroblotted onto a
polyvinylidene difluoride membrane. The membrane was probed with
anti-CAT and anti--lactamase rabbit antibodies, and the signals were
detected with alkaline phosphatase-conjugated goat anti-rabbit
immunoglobulin G by using BCIP (5-bromo-4-chloro-3-indolylphosphate)
and nitroblue tetrazolium (17).
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FIG. 5.
Northern blot analysis. Total tRNA from various
transformants (trmA) was isolated under acidic
conditions, separated on acid urea gels, and electroblotted onto a
Nytran membrane. The blots were hybridized to 5'
32P-labeled oligonucleotides complementary to positions 29 to 47 of the tRNA2fMet (U35A36) and to
positions 2 to 44 of E. coli tRNATyr
(26).
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To ensure that the
trmA strains were true to their
genotypes, we carried out modified base analysis of the
formylation-defective
tRNAs (Fig.
6). The tRNAs isolated
from the wild-type (
trmA+) background
contained rT modification (Fig.
6b and c, as shown
by arrows), whereas
those isolated from the
trmA strains lacked
this
modification (Fig.
6d to g, compare with Fig.
6b and c).
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FIG. 6.
Modified base analysis. Fresh overnight cultures
of the various transformants were subcultured (1% inoculum) in 5 ml of
2YT medium containing ampicillin (100 µg/ml) and kanamycin (25 µg/ml) and grown at 37°C with constant aeration for 4 h. The
cells were collected by centrifugation, suspended in 1 ml of
low-phosphate medium (20), supplemented with 500 µCi of
[32P]orthophosphate, and incubated at 37°C for 1 h. Initiator tRNA was purified from total tRNA preparation by
electrophoresis on a 15% preparative polyacrylamide gel under
nondenaturing conditions. The 32P-labeled tRNA (20,000 cpm)
were treated with nuclease P1 (1 µg) in 20 µl of 50 mM ammonium
acetate buffer (pH 5.3) at 37°C for 5 h, mixed with similarly
digested yeast total RNA (5 µg), and vacuum dried. Traces of ammonium
acetate were eliminated by vacuum drying the contents twice more after
dissolving them in 20 µl of water each time. The contents were
finally taken up in 2 µl of water and applied onto cellulose
thin-layer plates. Two-dimensional chromatography was performed by
using isobutyric acid-water-ammonium hydroxide in a 66:33:1
(vol/vol/vol) ratio as the solvent for the first dimension and 0.1 M
sodium phosphate (pH 7.2)-ammonium sulfate-n-propanol
in a 100:60:2 (vol/wt/vol) ratio as the solvent for the second
dimension exactly as described previously (21). The panels
show a standard pattern (a) or a modified base analysis of U35A36 (b,
d, and f) or G72G73/U35A36 (c, e, and g) tRNAs from KL16 (b and c),
KL356 (d and e), and G11-5-18 (f and g) strains. The position of rT is
indicated by arrows in panels b and c. rT is absent in panels d to e.
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Thus, our results clearly show that even though the
formylation-defective tRNAs (G72/U35A36 and G72G73/U35A36) accumulated
in the aminoacylated form and lacked rT modification, they failed
to
initiate protein synthesis. The inability of the
trmA
strains
to accrue any growth advantage in the presence of folate
biosynthesis
inhibitors on the one hand, and the failure to support
initiation
with the formylation-defective initiator tRNAs on the other,
shows
that the absence of rT modification is not sufficient for
formylation-independent
initiation.
Recently, formylation-independent initiation was effected in
E. coli (
8,
14),
Pseudomonas aeruginosa
(
14) and yeast
mitochondria (
12) by
disruption of the formylase gene. Although
the modification status of
the initiator tRNA was not analyzed
in any of these studies, the fact
that the methyltransferase gene
responsible for the conversion of U54
to T54 was not mutated suggested
that the rT modification occurred in
the initiator tRNA (
12).
Thus, taken together, these
studies suggest that requirement of
the lack of rT modification in the
initiators is neither absolute
nor sufficient for
formylation-independent initiation of protein
synthesis in
E. coli. Our interpretation, however, does not rule
out a possible
role of the lack of rT modification in formylation-independent
initiation in conjunction with mutations at some other location(s)
in
the
E. coli chromosome. In fact, this may well explain the
phenotype of the
E. coli mutant that initiated independent
of
formylation and showed partial lack of rT modification
(
3).
| |
ACKNOWLEDGMENTS |
This work was supported by a research grant from the Department of
Science and Technology and the Department of Biotechnology, New Delhi.
S.T. was a K. S. Krishnan senior fellow, and T.K.D. is supported
by a postdoctoral fellowship of the Department of Biotechnology.
S.T. and T.K.D. contributed equally to this study.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India. Phone: 91-80-309-2686. Fax: 91-80-360-2697. E-mail: varshney{at}mcbl.iisc.ernet.in.
| |
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Journal of Bacteriology, December 2001, p. 7397-7402, Vol. 183, No. 24
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.24.7397-7402.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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