From the Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received for publication, September 7, 2000, and in revised form, October 30, 2000
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ABSTRACT |
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The mode of recognition of tRNAs by
aminoacyl-tRNA synthetases and translation factors is largely unknown
in archaebacteria. To study this process, we have cloned the wild type
initiator tRNA gene from the moderate halophilic archaebacterium
Haloferax volcanii and mutants derived from it into a
plasmid capable of expressing the tRNA in these cells. Analysis of
tRNAs in vivo show that the initiator tRNA is aminoacylated
but is not formylated in H. volcanii. This result provides
direct support for the notion that protein synthesis in archaebacteria
is initiated with methionine and not with formylmethionine. We have
analyzed the effect of two different mutations (CAU The sequence and/or structural determinants in the anticodon and
in the acceptor stem of tRNAs play an important role in discrimination among tRNAs by aminoacyl-tRNA synthetases. The crystal structure analysis of several aminoacyl-tRNA synthetase-tRNA complexes from eubacteria and eukarya has provided a substantial amount of information on the molecular details of interactions involving these determinants (1-5). However, little is known either at the biochemical level or at
the structural level on interaction between aminoacyl-tRNA synthetases
and tRNA in archaea. The results of recent work, spurred by a knowledge
of the complete genome sequences of several archaea, have highlighted
some interesting and surprising differences between three archaeal
aminoacyl-tRNA synthetases and their eubacterial and eukaryal
counterparts. First, in contrast to lysyl-tRNA synthetases from
eubacteria and eukarya, which in general belong to Class II (5),
lysyl-tRNA synthetase from Methanococcus jannaschii belongs
to Class I (6, 7). Second, a single polypeptide of M. jannaschii has the activity (8, 9) of both a cysteinyl-tRNA synthetase and prolyl-tRNA synthetase. Third, the M. jannaschii tyrosyl-tRNA synthetase has a truncated C-terminal
region and lacks most of the tRNA anticodon binding region seen in
tyrosyl-tRNA synthetase from eubacteria and eukarya. This finding has
led to the suggestion (10, 11) that the anticodon of the M. jannaschii tyrosine tRNA is less important for aminoacylation
compared with the anticodon of tRNATyr from eubacteria and eukarya.
In view of the differences found in the three aminoacyl-tRNA
synthetases from M. jannaschii, it is important to study the mode of recognition of tRNAs in general by aminoacyl-tRNA synthetases in these and other archaeal systems. Here, we have studied this process
in vivo using the halophilic archaeon Haloferax
volcanii. We show that the anticodon sequence in the tRNA plays an
important role in the recognition of tRNAs by at least two of the
aminoacyl-tRNA synthetases, the methionyl-tRNA synthetase
(MetRS)1 and the valyl-tRNA
synthetase (ValRS). Our results also show that the initiator tRNA is
aminoacylated but is not formylated in H. volcanii. This
finding provides direct support for the commonly held notion that
protein synthesis in archaebacteria does not require formylation of the
initiator tRNA (12-14).
Strain, Plasmids, and Extracts--
The strain H. volcanii WFD11 and the plasmids pUCsptProM and pWL201 (15, 16)
were kindly provided by Drs. John R. Palmer and Charles J. Daniels,
Department of Microbiology, The Ohio State University, Columbus, OH.
The Escherichia coli strains used in this work are E. coli XL1-blue (recA1 endA1 gyr96 thi-1 hsdR17 supE44 relA1
lac (F' proAB lacIqZ Isolation of H. volcanii Genomic DNA--
H. volcanii WFD11 was
grown in a medium containing the following components per liter:
125 g of NaCl, 45 g of MgCl2·6H2O, 10 g of MgSO4·7H2O, 10 g of KCl,
1.34 g of CaCl2·2H2O, 3 g of yeast
extract, 5 g of tryptone (17). Cells from a 30-ml culture (grown
for 48 h with 4% inoculum at 37 °C with aeration) were harvested and suspended in 9.0 ml of 50 mM Tris-HCl (pH
8.0), 25 mM Na2EDTA by vortexing. Significant
lysis was observed during this step, and lysis was taken to completion
with the addition of SDS to a final concentration of 1%. The lysate
was extracted with an equal volume of phenol (saturated with 100 mM Tris-HCl (pH 8.0)) by gentle mixing (inversions in a
cyclo mixer at low rpm for 15 min at room temperature). The
aqueous phase was collected and reextracted with phenol once more and
then twice with chloroform:isoamylalcohol (24:1). The aqueous phase was
collected, and sodium acetate (pH 5.4) was added to a final
concentration of 0.3 M followed by addition of chilled
ethanol. The genomic DNA was spooled, as it precipitated, onto a
sterile glass rod, rinsed with 70% ethanol, air dried, and gently
suspended in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA
(TE) buffer.
Cloning of the Initiator tRNA Gene--
Based on the sequence of
the tRNAiMetlocus provided by Dr.
C. J. Daniels, the following primers that contained the sites for XbaI and BamHI (italicized) were designed for
polymerase chain reaction amplification of the
tRNAiMet gene: HvMiF1,
5'-ACATTCTAGACTGTTTGTTGATTCAGCGGGA-3'; HvMiR1, 5'-CTCGGGATCCGGAGTTGAGGTCGGCAAACCA-3'. The primers (100 pmol
each) were used to amplify the tRNAiMet
gene using H. volcanii genomic DNA as template under the
following conditions: 95 °C/1-min initial denaturation followed by
20 cycles, with each cycle containing the steps 95 °C/1 min,
50 °C/1 min, and 72 °C/1 min. This was followed by incubating the
tubes at 72 °C for 5 min. The purified polymerase chain reaction
product was digested with XbaI and BamHI and
cloned into the plasmid pUCsptProM at these sites to generate
pUCsptHvMeti. The mutations in the anticodon of the tRNA
(U35A36 and G34C36) were generated using pUCsptHvMeti as template by the Quik Change mutagenesis
procedure. The smaller HindIII+EcoRI fragment
containing the initiator tRNA gene and its mutants from the
pUCsptHvMeti plasmid was subcloned into the shuttle plasmid
pWL201 at these sites to generate pWL201HvMeti, pWL201HvMetiU35A36, and
pWL201HvMetiG34C36, respectively. All initial cloning
was done in E. coli XL-1 blue. After confirming the sequence
of the desired wild type and mutant initiator tRNA genes, the
respective plasmids were used to transform E. coli GM2163.
The plasmid DNA isolated from this strain (lacking adenine methylation)
was then used to transform H. volcanii.
Transformation of H. volcanii--
The protocol was adapted from
Cline et al. (18). All operations were at room temperature.
Cells from a 50-ml culture grown to an A600
nm of ~1.0 (25 h at 37 °C) were harvested and gently suspended in 5 ml of spheroplasting buffer (50 mM Tris-HCl
(pH 8.3), 0.8 M NaCl, 30 mM KCl, 15% sucrose,
15% glycerol). To 200 µl of the cell suspension, 20 µl of 0.5 M Na2EDTA (pH 8.0) was added and gently mixed.
Plasmid DNA (1 µg) in 20 µl of spheroplasting buffer was added and
mixed gently. An equal volume (240 µl) of polyethylene glycol
solution (60% polyethylene glycol-600 w/v in spheroplasting buffer)
was then added, and the mixture was gently mixed and incubated for 20 min at room temperature. The cells were diluted by adding 9 ml of
spheroplast dilution buffer (50 mM Tris-HCl (pH 7.2), 3.4 M NaCl, 175 mM MgSO4, 30 mM KCl, 5 mM CaCl2, 15% sucrose),
harvested by centrifugation, and resuspended in 1 ml of a 1:1 mixture
of spheroplast dilution buffer and H. volcanii growth
medium. The transformants were allowed to recover at 37 °C
with mild aeration for 12 h. Appropriate aliquots were mixed with
molten top agar (55 °C) and poured onto agar plates. The contents of
the plates were as follows: bottom agar (per liter), 50 mmol
Tris-HCl (pH 7.2), 180 g of NaCl, 43 g of MgSO4,
2.5 g of KCl, 0.7 g of CaCl2·2H2O,
3 g of yeast extract, 5 g of tryptone, and 15 g of agar;
top agar, same as above except for 8 g/liter agar and 4 mg/liter
mevinolin. Mevinolin was converted to the sodium salt as described
(19). The plates were incubated at 37 °C for 7 days.
Isolation of Total tRNA under Acidic Conditions--
The
mevinolin-resistant transformants of H. volcanii were grown
in liquid medium in the presence of mevinolin (4 mg/liter) for 3 days. The cells from the 3.0-ml culture were chilled on wet ice. All
subsequent steps were carried out in the cold. The cells were
harvested, resuspended in 0.3 ml of 0.3 M NaOAc (pH 4.5)
and 10 mM Na2EDTA, and subjected to two
extractions (1 min each) with equal volumes of phenol equilibrated with
the same buffer. Total nucleic acids were recovered from the aqueous
phase by mixing with 2.5 volumes of ethanol and centrifugation. The pellet was washed with 70% ethanol and dissolved in 20 µl of 10 mM NaOAc (pH 4.5), 1 mM
Na2EDTA.
Detection of tRNAs by Northern Blotting--
tRNA (0.1 A260 unit) was subjected to electrophoresis on
acid-urea polyacrylamide gels and electroblotted onto a Nytran-Plus membrane (Schleicher & Schuell) as described (20). The membrane was
washed with 4× SET (1× SET: 0.05 M NaCl, 0.03 M Tris-HCl, 2 mM Na2EDTA (pH 8.0)
containing 1% SDS), baked at 70 °C for 90 min, and prehybridized at
42 °C in 4× SET containing 250 µg/ml sheared salmon sperm DNA,
1% SDS, and 10× Denhardt's solution (1× Denhardt's solution:
0.02% polyvinylpyrrolidone 40, 0.02% bovine serum albumin, and 0.02%
Ficoll). tRNAs were detected by hybridization to 5'-32P
labeled oligonucleotide probes complementary to nucleotides 29-47 of
the wild type and mutant initiator tRNAs, 26-c5 of H. volcanii tRNAGGASer, and
34-47 of H. volcanii
tRNAGACVal (21, 22). Hybridization was
performed in the same solution at 42 °C for 12 h. The membrane
was washed twice (20 min each) with 3× SET and 0.2% SDS at 42 °C.
For detecting the mutant tRNAs, the blot was washed with 0.75× SET,
0.05% SDS at 55 °C for 20 min. This wash eliminated the signal from
the probe hybridizing to the wild type initiator
tRNAiMet.
Measurement of Rates of Deacylation of
Aminoacyl-tRNAs--
Total tRNA was prepared from H. volcanii/pWL201HvMetiG34C36 under acidic conditions as
described above. About 2 A260 units of tRNA in
10 µl were treated with 0.2 M Tris-HCl (pH 9.6). Aliquots (1 µl) were withdrawn after 15, 30, 60, 90, 120, and 180 min, mixed
with 2 µl of 0.3 M NaOAc (pH 4.5), and snap-frozen on dry ice (23). At the end of all the time points, the volume was made up to
10 µl with 10 mM NaOAc (pH 4.5). For analyzing the deacylation of Met-tRNAiMet, the
experimental conditions were the same except that incubation times were
shorter: 2.5, 5, 7.5, 10, 15, and 30 min. An aliquot corresponding to
0.08 A260 unit of tRNA from each time point was analyzed by acid-urea polyacrylamide gel electrophoresis, and the tRNAs
were detected by Northern blot analysis using appropriate hybridization
probes as described above.
Isolation of Total tRNAs--
Total tRNAs were prepared from 900 ml of H. volcanii harboring the pWL201 series of plasmids
containing the respective tRNA genes. The cells were grown for 60 h in the presence of 4 mg/liter mevinolin. The cells were harvested,
resuspended first in 4.5 ml of 3 M NaOAc (pH 5.4), and then
made up to 40 ml with TE buffer. The suspension was extracted with an
equal volume of phenol saturated with the same buffer by shaking at
room temperature for 20 min. The aqueous phase was collected and mixed
with 2.5 volumes of ethanol, and the nucleic acids were recovered by
centrifugation. The pellet was washed with 70% ethanol, dried, and
resuspended in 12 ml of the TE buffer. High molecular weight nucleic
acids were precipitated by adding 3 ml of 5 M NaCl. Total
tRNA was recovered from the supernatant by ethanol precipitation. The
pellet was washed with 70% ethanol, dried, and resuspended in 15 ml of
TE buffer containing 0.1 M NaCl. The tRNAs were further
purified by DEAE-cellulose chromatography (24).
In Vitro Aminoacylation and Formylation of tRNA--
Total tRNA
(0.6 A260 unit) prepared from
H.volcanii/pWL201HvMeti cells as described above
was aminoacylated with methionine using E. coli MetRS or was
aminoacylated with methionine and subsequently formylated using
E. coli methionyl-tRNA formyltransferase (MTF). The
reaction mixture was extracted with phenol equilibrated with 10 mM NaOAc (pH 4.5). The tRNA was precipitated and analyzed
on acid-urea polyacrylamide gels and detected by Northern blot
hybridization. Total tRNA prepared from
H.volcanii/pWL201HvMetiU35A36 cells was aminoacylated with glutamine using an S-100 extract prepared from E. coli BL21(DE3)/pET3-glnS, which overproduces
E. coli glutaminyl-tRNA synthetase (GlnRS) (a kind gift from
Dr. M. Ibba).
The H. volcanii Initiator tRNA--
In the absence of an archaeal
in vitro protein synthesis system that can translate a
natural mRNA or in the absence of any genetic analysis involving
components of the translational initiation machinery, identification of
the initiator tRNAiMet in halobacteria
is based on the ability of the tRNA to be aminoacylated with E. coli MetRS and to be formylated by the E. coli MTF and the ability of the fMet-tRNAiMet to
initiate protein synthesis in an E. coli cell-free system (24). These were also the criteria used to identify the initiator tRNA
species in eukarya such as yeast (25), mammalian cells (26), and fungal
mitochondria (27) prior to their establishment as initiator tRNAs in
eukaryal in vitro protein synthesis systems. Based on these
criteria, the sequence of initiator tRNA and/or the gene for the
initiator tRNA is known from the following nine archaea:
Archaeoglobus fulgidus (GenBankTM accession
number NC000917), Halococcus morrhuae (28), H. volcanii (21), Methanobacterium thermoautotropicum
(GenBankTM accession number NC000916), M. jannaschii (GenBankTM accession number
NC000909), Pyrococcus abyssi (GenBankTM
accession number AL096836), Pyrococcus horikoshii
(GenBankTM accession number NC000961), Sulfolobus
acidocaldarius (28), and Thermoplasma acidophilum
(28).
Plasmid-directed Expression in Vivo of the H. volcanii Initiator
tRNA Gene--
To study the effect, in vivo, of mutations
in the anticodon sequence of initiator tRNA, we cloned the initiator
tRNA gene of H. volcanii in the plasmid pWL201 (15, 16).
This shuttle vector contains a bla gene coding for
resistance to ampicillin, an ori for maintenance of the
plasmid in E. coli, another ori for maintenance
of the plasmid in H. volcanii, and a gene coding for a
mutant 3-hydroxy-3-methylglutaryl-CoA reductase, which confers resistance of H. volcanii to mevinolin. The expression of
the initiator tRNA in the resulting plasmid pWL201HvMeti is
driven by the H. volcanii tRNALys promoter. This
plasmid was isolated from E. coli XL1-blue strain and used
to transform H. volcanii WFD11, and the transformants were selected in the presence of mevinolin. To establish the presence of plasmids in the mevinolin-resistant transformants, an attempt was
made to isolate plasmid DNA from these cells. The preparation failed to
yield ampicillin-resistant transformants when introduced into
the E. coli XL1-blue strain. Plasmid DNA was also not
detected in this preparation upon agarose gel electrophoresis and
ethidium bromide staining. These results suggest that the
mevinolin-resistant transformants of H. volcanii do not
contain the plasmids bearing the marker
3-hydroxy-3-methylglutaryl-CoA reductase gene and that the
resistance is most likely due to integration of the plasmid by
homologous recombination of the mutant
3-hydroxy-3-methylglutaryl-CoA reductase gene with the
chromosomal gene. Similar observations were made by Holmes et
al. (29), who reported the existence of a restriction barrier
between E. coli and H. volcanii. This restriction
can be avoided by using DNA lacking the
N6-methyl adenosine modification within the GATC
sequences. For this purpose, the pWL201HvMeti plasmid was
used to transform the dam Initiator tRNA Is Not Formylated in H. volcanii--
A feature
unique to all initiator tRNAs is the presence of three consecutive G:C
base pairs in the anticodon stem (Fig. 1; also see Ref. 14 for a review). Other features unique to eubacterial initiator tRNAs include a mismatch between positions 1 and 72 at the
end of the acceptor stem and a R11:Y24
base pair in the D-stem (30). Features unique to the eukaryal initiator
tRNAs include a A1:U72 base pair at the end of
the acceptor stem and A54 and A60 in the
T-loop. The archaeal initiator tRNAs have an
A1:U72 base pair at the end of the acceptor
stem found in the eukaryal initiator tRNAs and a
R11:Y24 base pair in the D-stem found in the
eubacterial initiator tRNAs (2, 28).
Initiator tRNA is present predominantly as fMet-tRNAfMet in
eubacteria and as Met-tRNAiMet in
eukarya. Two of the unique features of eubacterial initiator tRNAs, the
1x72 mismatch and the R11:Y24 base pair, play a
role in formylation of the E. coli initiator Met-tRNA by
E. coli MTF (31-33). Previous studies showed that extracts of Halobacterium cutirubrum, an extreme halophile, lacks MTF
activity. Furthermore, tRNA isolated from
[35S]methionine-labeled halobacterial cells contained
Met-tRNA but no fMet-tRNA (12). These findings, combined with the more
recent finding from the genome sequence of several archaea
(GenBankTM accession numbers NC000917, NC000916, NC000909,
AL096836, and NC000961) that they lack a protein with homology to
eubacterial MTFs, have led to the widespread notion that archaea
initiate protein synthesis without formylation of the initiator tRNA.
Here, we have investigated directly the in vivo state of the
H. volcanii initiator tRNA with a probe specific to this
tRNA (Fig. 2). For use as markers, total
tRNA isolated from these cells was aminoacylated in vitro
with E. coli MetRS (Fig. 2A, lane
2) or aminoacylated with MetRS and subsequently formylated with
E. coli MTF (lane 3) and subjected to
electrophoresis on the same gel. As for the E. coli
initiator tRNAfMet (20), all three forms of the H. volcanii initiator tRNA, the tRNAiMet, the
Met-tRNAiMet, and the
fMet-tRNAiMet, are separated clearly
from one another (Fig. 2A, lanes 1, 2, and 3, respectively). The mobility of H. volcanii initiator tRNA isolated from the cells under acidic
conditions (Fig. 2A, lane 5) shows that
the tRNAiMet is quantitatively
aminoacylated; however, there was no detectable accumulation of any
fMet-tRNAiMet species. This result
provides direct support to the notion that protein synthesis in
halobacteria is initiated with methionine and not with
formylmethionine.
Fig. 2B, lane 2 shows that the endogenous
H. volcanii initiator tRNA was also not formylated in
vivo. Thus, the lack of formylation of the H. volcanii
initiator tRNA (Fig. 2A, lane 5) is not due to a
substantial overproduction of the initiator tRNA in cells carrying the
pWL201HvMeti plasmid overwhelming the limiting amounts of a putative
Met-tRNAi formyltransferase activity in H. volcanii. Further evidence for this is also derived from the
results of a Northern blot analysis similar to that in Fig.
2B, lanes 2 and 4, which show that the
initiator tRNA was overproduced only to the extent of 50% in H. volcanii cells carrying the pWL201HvMeti plasmid over
the endogenous initiator tRNA.
The CUA Anticodon Mutant Is Aminoacylated Extremely Poorly in
Vivo--
The plasmid pWL201HvMetiU35A36 containing the
U35A36 anticodon sequence mutant initiator tRNA gene
(tRNAiMet (CUA)) was used to transform
H. volcanii. The transformants were analyzed for the
expression of tRNA by acid-urea polyacrylamide gel electrophoresis
followed by Northern blot analysis using a probe specific to the mutant
tRNA. As expected, a band corresponding to the U35A36 mutant
tRNAiMet was found only in cells
containing the plasmid pWL201HvMetiU35A36 (Fig.
3A, compare lanes 1 and 2 to lanes 3 and 4). However,
based on PhosphorImager analysis, only ~8% of the mutant tRNA
was aminoacylated in vivo (Fig. 3A,
lane 4). In contrast, the serine tRNA, which was used as an
internal control, was fully aminoacylated (Fig. 3A,
lanes 2 and 4). Thus, a two-nucleotide change in
the anticodon sequence of the H. volcanii initiator tRNA
results in a tRNA that is aminoacylated extremely poorly. These results
suggest that, as in eubacteria and eukarya, the anticodon sequence of a
tRNA is important for its aminoacylation by the H. volcanii
MetRS. Whether the residual aminoacylation of the mutant tRNA, to the extent of ~8%, is with methionine or another amino acid is not known.
In E. coli, the corresponding E. coli U35A36
mutant initiator tRNA is aminoacylated with glutamine by GlnRS (34,
35). To determine whether the U35A36 mutant
tRNAiMet expressed in H. volcanii can be aminoacylated with glutamine in vitro,
we isolated tRNAs from the cells transformed with the plasmid
pWL201HvMetiU35A36. The total tRNA preparation was
aminoacylated with glutamine using E. coli GlnRS, and the
products were analyzed by acid-urea polyacrylamide gel electrophoresis
followed by Northern blot analysis (Fig. 3B). The results
show that the U35A36 mutant tRNAiMet can
be aminoacylated in vitro by the E. coli GlnRS
(Fig. 3B, lanes 2 and 3). Thus, the
lack of aminoacylation of the H. volcanii mutant initiator
tRNA in vivo is due to the absence of an aminoacyl-tRNA synthetase that recognizes this mutant initiator tRNA.
Archaea lack GlnRS and, like most Gram-positive eubacteria (36), use a
two-step pathway to generate Gln-tRNA (21, 37). This pathway (the
glutamyl-tRNA synthetase-glutamine amidotransferase pathway) involves
aminoacylation of the tRNA with glutamic acid followed by conversion of
the glutamic acid on the tRNA to glutamine using the enzyme glutamine
amidotransferase (38). The finding that the U35A36 mutant of the
H. volcanii initiator tRNA is mostly not aminoacylated
in vivo also suggests that the mutant tRNA is a poor
substrate for the H. volcanii glutamyl-tRNA synthetase.
The GAC Anticodon Mutant Is Most Likely Aminoacylated with
Valine--
A second anticodon mutant initiator tRNA gene (G34C36
mutant) containing the GAC anticodon was constructed, and the resulting plasmid pWL201HvMetiG34C36 was used to transform H. volcanii. Expression of the G34C36 mutant initiator tRNA
(tRNAiMet (GAC)) was analyzed by
acid-urea Northern blotting using an oligonucleotide probe specific to
the mutant tRNA. The G34C36 mutant tRNA was detected only in cells
containing the pWL201HvMetiG34C36 plasmid (Fig.
4, compare lanes 1 and
2 to lanes 3 and 4). Approximately 45% of the mutant initiator tRNA was aminoacylated in H. volcanii (Fig. 4, lane 4). The tRNA isolated
from the cells was deacylated with 0.25 M Tris-HCl (pH 9.6)
for 20 min at 37 °C (23). Whereas the H. volcanii
Ser-tRNASer was completely deacylated under these
conditions, up to 30% of the H. volcanii G34C36 mutant
tRNAiMet was still aminoacylated (Fig.
4, lane 3).
The very slow rate of deacylation of the G34C36 mutant aminoacyl-tRNA
suggests that the tRNA is not aminoacylated with methionine or most of
the other amino acids in H. volcanii (23, 39, 40). It is
known that the nature of the amino acid attached to the tRNA determines
the rate of deacylation of the aminoacyl-tRNA, with valine and
isoleucine having the slowest rates of deacylation (23, 40). The G34C36
mutant initiator tRNA has the anticodon sequence (GAC) corresponding to
that of valine tRNA, and it is known that in eubacteria and eukarya,
the anticodon sequence is important for aminoacylation of the tRNA with
valine (41). Therefore, it is most likely that the G34C36 mutant
initiator tRNA is aminoacylated with valine in H. volcanii.
To investigate this further, total tRNA isolated from H. volcanii/pWL201HvMetiG34C36 cells under acidic
conditions was subjected to deacylation in 0.2 M Tris-HCl (pH 9.6) at 37 °C. The time course of deacylation of the G34C36 mutant aminoacyl-tRNA was compared with those of the H. volcanii Met-tRNAiMet and
Val-tRNA1Val. Deacylation was followed
by withdrawing aliquots of the reaction mixture at intervals and
analyzing the samples by acid-urea gel electrophoresis followed by
Northern blot analysis using probes specific for the endogenous
tRNAiMet,
tRNA1Val, and the G34C36 mutant
tRNAiMet (23). The results are shown in
Fig. 5. A plot of the residual aminoacyl-tRNA versus time (Fig.
6), based on the PhosphorImager analysis of pixels in the tRNA and aminoacyl-tRNA bands, shows that the
half-lives of deacylation of the aminoacyl-tRNAs are ~7 min
(Met-tRNAiMet), ~52 min
(Val-tRNA1Val), and ~48 min (the
G34C36 mutant aminoacyl- tRNAiMet).
The extreme closeness of the half-life of the G34C36 mutant aminoacyl-tRNAiMet to that of
Val-tRNAVal indicates that the amino acid attached to the
tRNA is not methionine but, most likely, valine. These results suggest
that the anticodon sequence of a tRNA is also important for its
aminoacylation by the H. volcanii ValRS.
Further evidence that the G34C36 mutant initiator tRNA is aminoacylated
with valine was obtained by a comparison of the valine and isoleucine
acceptor activities of total tRNA isolated from cells expressing the
G34C36 mutant initiator tRNA. Fig. 7
shows a time course of aminoacylation of tRNAs with valine
(left) and with isoleucine (right) using
cell-free extracts of H. volcanii. Total tRNA isolated
from cells expressing the G34C36 mutant initiator tRNA shows an
increase in valine acceptor activity compared with the endogenous tRNA
(Fig. 7, left) but no increase in isoleucine acceptor
activity (Fig. 7, right) or in methionine acceptor activity (data not shown). These results highlight the importance of the anticodon in aminoacylation of the tRNA by H. volcanii
ValRS. The slower rate of aminoacylation of the G34C36 mutant initiator tRNA with valine compared with the endogenous tRNAVal
suggests that, whereas the anticodon sequence is important for aminoacylation of a tRNA by the H. volcanii ValRS, there are
other determinants that are also important and that are lacking in the G34C36 mutant initiator tRNA. This result is also consistent with the
finding that only ~45% of the G34C36 mutant initiator tRNA is
aminoacylated in H. volcanii (Fig. 4, lane
4).
Conclusion--
As a first step toward studying the role of the
anticodon sequence in aminoacylation of tRNAs by aminoacyl-tRNA
synthetases in an archaebacterium, we have described the
expression and analysis in vivo of two anticodon sequence
mutants of the H. volcanii initiator tRNA. We show that the
anticodon sequence is important for aminoacylation by at least two of
the H. volcanii aminoacyl-tRNA synthetases, the MetRS and
the ValRS. These results are similar to those for the corresponding
eubacterial and eukaryal enzymes. It will be interesting to extend
these studies to other anticodon sequence mutants to see whether the
relative importance of the anticodon sequence for aminoacylation of a
tRNA by the archaeal aminoacyl-tRNA synthetases is generally similar to
those of the corresponding eubacterial and eukaryal aminoacyl-tRNA synthetases.
CUA and
CAU
GAC) in the anticodon sequence of the initiator tRNA on its
recognition by the aminoacyl-tRNA synthetases in vivo. The
CAU
CUA mutant was not aminoacylated to any significant extent
in vivo, suggesting the importance of the anticodon in
aminoacylation of tRNA by methionyl-tRNA synthetase. This mutant
initiator tRNA can, however, be aminoacylated in vitro by
the Escherichia coli glutaminyl-tRNA synthetase,
suggesting that the lack of aminoacylation is due to the absence in
H. volcanii of a synthetase, which recognizes the mutant
tRNA. Archaebacteria lack glutaminyl-tRNA synthetase and utilize a
two-step pathway involving glutamyl-tRNA synthetase and glutamine
amidotransferase to generate glutaminyl-tRNA. The lack of
aminoacylation of the mutant tRNA indicates that this mutant tRNA is
not a substrate for the H. volcanii glutamyl-tRNA
synthetase. The CAU
GAC anticodon mutant is most likely aminoacylated
with valine in vivo. Thus, the anticodon plays an important
role in the recognition of tRNA by at least two of the halobacterial
aminoacyl-tRNA synthetases.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
M15
Tn10 (Tetr))) and E. coli
GM2163 (F
ara-14 leuB6 thi-1 fhuA31
lacY1 tsc-78 galK2 galT22 supE44 hisG4 rpsL136
(Strr) xyl-5 mtl-1 dam14::Tn9
(Camr) dcm-6 mcrB1 hsdR2
(rk
mk+) mcrA).
H. volcanii S40 extract was a kind gift of Dr. Mechthild Pohlschröeder, University of Pennsylvania, Philadelphia, PA.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
E. coli
GM2163 strain, which is deficient in the
N6-adenine methyl transferase. When H. volcanii cells were transformed with the plasmid isolated from the
dam
E. coli strain, the
efficiency of transformation improved by at least one order of
magnitude. The mevinolin-resistant transformants could be maintained
and subcultured repeatedly without loss of the plasmid. The plasmid
isolated from these transformants also yielded
ampicillin-resistant colonies when introduced into E. coli XL1-blue. Based on these results suggesting the stable
maintenance of plasmids containing the wild type initiator tRNA gene in
H. volcanii, we generated the constructs containing the
U35A36 and G34C36 mutations in the anticodon sequence of the initiator tRNA.
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Fig. 1.
Top, schematic diagram of eubacterial
and eukaryal initiator tRNAs. The unique features in the eubacterial
(left) and eukaryal (right) initiator tRNAs are
highlighted by arrows. Bottom, sequence of the
H. volcanii initiator tRNA in cloverleaf form. The mutations
introduced into the anticodon sequence CAU (boxed)
are indicated by arrows emanating from it. Also highlighted
(arrows) are features common to archaeal initiator
tRNAs.
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Fig. 2.
RNA blot hybridization of tRNA isolated from
H. volcanii cells. A, total tRNA isolated from
H. volcanii/pWL201 HvMeti under acidic
conditions was subjected to acid-urea polyacrylamide gel
electrophoresis with or without alkali treatment (lanes 4 and 5) and electroblotted onto a membrane. Lanes
1-3 provide markers of tRNAiMet,
Met-tRNAiMet, and
fMet-tRNAiMet, respectively (details are
given under "Experimental Procedures"). The
tRNAiMet was detected using a probe
directed against nucleotides 29-47 of the tRNA. B, total
tRNA isolated from H. volcanii/pWL201 or
pWL201HvMeti under acidic conditions was used with
(lanes 1 and 3) or without (lanes 2 and 4) alkali treatment. The
tRNAiMet and tRNASer were
detected using probes directed against nucleotides 29-47 of
tRNAiMet and nucleotides 26-c5 of
H. volcanii tRNASer, respectively (21,
22).
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Fig. 3.
RNA blot hybridization of tRNAs isolated from
H. volcanii carrying the
pWL201HvMetiU35A36 plasmid. A, analysis of
tRNAs isolated under acidic conditions from H. volcanii
pWL201, the empty vector (lanes 1 and 2), and
H. volcanii pWL201 HvMetiU35A36 (lanes
3 and 4). B, analysis of the H. volcanii U35A36 mutant initiator tRNA before and after
aminoacylation with E. coli GlnRS in vitro. The
tRNAs were separated by acid-urea polyacrylamide gel electrophoresis
and detected by using probes specific for the H. volcanii
U35A36 mutant initiator tRNA and the tRNASer, used as a
control. Details are given under "Experimental
Procedures."aa, aminoacyl.
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Fig. 4.
Chemical deacylation of the aminoacylated
form of H. volcanii G34C36 mutant initiator tRNA
and Ser-tRNA. The reaction was followed by acid-urea
polyacrylamide gel electrophoresis and RNA blot hybridization with the
tRNA sequence-specific DNA probes. Details are given under
"Experimental Procedures."aa, aminoacyl.
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Fig. 5.
Comparison of rates of chemical deacylation
of aminoacylated forms of the G34C36 mutant initiator tRNA
(A) and the wild type initiator tRNA
(B) as followed by Northern blot analysis with tRNA
sequence-specific probes. aa,
aminoacyl.
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Fig. 6.
Quantitation of the data in Fig. 5 by
PhosphorImager analysis of the pixels in the tRNA and
aminoacyl-tRNA bands. Plot of residual aminoacyl-tRNA
versus time of incubation. The data for the endogenous
Val-tRNAVal (GAC) was obtained from the same filter as used
for panel A of Fig. 5 after stripping the probe specific for
the G34C36 mutant initiator tRNA and reprobing with an oligonucleotide
specific for the H. volcanii
tRNAVal. aa, aminoacyl.
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Fig. 7.
Time course of aminoacylation with valine
(left) and isoleucine (right) of
H. volcanii total tRNA isolated from cells carrying
the empty vector pWL201 or pWL201HvMetiG34C36. The
incubation mixture contained 1 A260 unit of
total tRNA and 105 µg of proteins in an H. volcanii S40
extract.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Charles Daniels for the H. volcanii strains and vectors, Dr. Mechthild Pohlschröeder for the H. volcanii S40 extracts, and Drs. Michael Ibba and Dieter Söll for E. coli GlnRS. We thank Annmarie McInnis for patience and care in the preparation of this manuscript.
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FOOTNOTES |
---|
* This work was supported by Grant R37GM17151 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
617-253-4702; Fax: 617-252-1556; E-mail: bhandary@mit.edu.
Published, JBC Papers in Press, October 31, 2000, DOI 10.1074/jbc.M008206200
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ABBREVIATIONS |
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The abbreviations used are: MetRS, methionyl-tRNA synthetase; ValRS, valyl-tRNA synthetase; MTF, methionyl-tRNA formyltransferase; GlnRS, glutaminyl-tRNA synthetase.
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