Nucleic Acids Research, 1993, Vol. 21, No. 6 1335-1338
Lack of peptide-release activity responding to codon UGA
in Mycoplasma capricolum
Yuji Inagaki*, Yoshitaka Bessho and Syozo Osawa+
Laboratory of Molecular Genetics, Department of Biology, School of Science, Nagoya University,
Chikusa-ku, Nagoya 464-01, Japan
Received January 29, 1993; Accepted February 18, 1993
ABSTRACT
In Mycoplasma capricolum, a relative of Gram-positive
eubacteria with a high genomic AT-content (75%),
codon UGA is assigned to tryptophan instead of
termination signal. Thus, in this bacterium the release
factor 2 (RF-2), that recognizes UAA and UGA
termination codons in eubacteria such as Escherichia
coli and Bacillus subtilis, would be either specific to
UAA or deleted. To test this, we have constructed a
cell-free translation system using synthetic mRNA
including codon UAA [mRNA(UAA)], UAG [mRNA(UAG)] and UGA [mRNA(UGA)] in-frame. In the absence of
tryptophan, the translation of mRNA(UGA) ceased at
UGA sites without appreciable release of the
synthesized peptides from the ribosomes, whereas with
mRNA(UAA) or mRNA(UAG) the bulk of the peptides
was released. Upon addition of the E.coil S-100 fraction
or B.subtilis S-100 fraction to the translation system,
the synthesized peptides with mRNA(UGA) were almost
completely released from the ribosomes, presumably
because of the presence of RF-2 active to UGA in the
added S-100 fraction. These data suggest that RF-2 is
deleted or its activity to UGA is strongly weakened in
M.capricolum.
To clarify this, a cell-free system of M. capricolum was
constructed for translation of the added synthetic mRNAs
containing UAA, UAG or UGA in-frame. When tryptophan was
removed from the system, translation ceased just before UGA
and the bulk of the synthesized peptides was attached to the
ribosomes. The peptides were released upon incubation with
puromycin or the E.coli or B.subtilis S-100 fraction. These results
indicate that UGA-dependent RF-activity is absent or at least very
weak.
MATERIALS AND METHODS
INTRODUCTION
Cell culture
Mycoplasma capricolum [American Type Culture Collection
27343 (Kid)] cells were grown as described (10) and stored at
-0°C until use.
Escherichia coli K12 JCH1 11 (F-, met-, trp-) and Bacillus
subtilis BD170 (thr-, trp-) cells were grown in the LB medium
until middle log phase at 37°C with shaking. Cells were collected
by centrifugation at 8,000xg at 0°C and washed twice with M9
medium (0.6% Na2HPO4/0.3 % KH2PO4/0.5 % NaCl/0. 1%
NH4Cl/2mM MgSO4 /O.lmM CaCl2/0.2% glucose). E.coli or
B.subtilis cells were subjected to tryptophan starvation by shaking
in M9 medium including methionine (100/lg/ml) or threonine
(10Og/ml) at 37°C for 3 hrs, collected and stored at -70°C
until use.
UAA, UAG and UGA are termination codons in the universal
genetic code. In eubacteria such as Escherichia coli (1,2) and
Bacillus subtilis (3), two protein factors, release factor 1 (RF-1)
and release factor 2 (RF-2), participate in the recognition of the
termination codons and in release of nascent polypeptides from
the ribosomes. RF-1 recognizes UAA and UAG, whereas RF-2
recognizes UAA and UGA (1).
It is well established that a bacterium M. capricolum uses UGA as a tryptophan codon instead of termination signal (4-7).
Codon UGA is translated by tRNATUA. It is then expected that
M. capricolum does not possess the UGA-dependent RF-activity
in response to the use of UGA as a regular tryptophan codon.
Preparation of the S-30 fraction and S-100 fraction
The M. capricolum S-30 fraction was prepared as described (8),
and was dialyzed against 2 1 of buffer A (1OmM Tris-HCl
pH7.8/lOmM magnesium acetate/60mM NH4CI), with changes
at 1 hr-interval for 6 hrs to remove amino acids and stored at
-70°C until use. The E.coli and B.subtilis S-100 fractions were
prepared as described (9), dialyzed against 2 1 of buffer A with
changes at 1 hr-interval for 8 hrs and stored at -70°C until use.
The final protein concentration of the S-30 fraction was
9.8mg/ml, and those of the E. coli and B.subtilis S-100 fraction
were 9.0mg/ml and 3.0mg/ml, respectively.
To whom correspondence should be addressed
'Present address: Cosmo Annex ID, 7 Umezono,
Osaka 569, Japan
*
Hiroji-cho, Showa-ku, Nagoya 466, Japan. After June 1993: Biohistory Research Hall, Takatsuki-shi,
1336 Nucleic Acids Research, 1993, Vol. 21, No. 6
Preparation of synthetic messenger RNAs
The fragments having sequences corresponding to mRNAs with
restriction enzyme sites (Pst I and Sac I) for cloning were
synthesized by Gene Assembler Plus (Pharmacia LKB) (Fig. 1).
The synthesized inserts were ligated to plasmid vector Bluescript
II KS+ (stratagene) at the Pst I and Sac I sites. The plasmids
were transformed to E.coli DHIOB. The plasmids carrying
correct insert were selected by DNA sequencing. The
transcription of mRNA from DNA with the T7 RNA polymerase
was done as described (10). All enzymes used were purchased
from Takara Shuzo Co., LTD.
Cell-free translation
The standard reaction mixture contained, in 90p1, 50mM Tris-Cl
pH7.8, 3.75mM of magnesium acetate, 60rnM of NH4Cl, lmM
of dithiotheitol, 5i4M of each of methionine and tryptophan,
666KBq of [3H]isoleucine (3.74TBq/mmol, Amersham), 7,ug of
synthetic mRNA and 27,ad of the S-30 fraction. In some
experiments, 16t1 of the M. capricolum S-30 fraction were used
with addition of 16yd the B.subtilis S-100 fraction or 5,1i of the
E.coli S-100 fraction. The reaction mixture was incubated at
37°C. Ten gl aliquot was taken out at intervals and was treated
with 1N NaOH at 370C for 15 min, followed by 20% (wt/vol)
trichloroacetic acid (TCA) treatment at 0°C for 30 min. The
TCA-insoluble material was collected on glass filters (Whatman
GF/C) and thoroughly washed with cold 10% TCA. The
radioactivity was measured by liquid scintillation counter
(Tri-Carb).
Sucrose-gradient centrifugation
Ninety ,ul of the reaction mixture were incubated at 37°C for
10 min. When lmM puromycin was added, the mixture was
further incubated at 370C for 10 min. It was then layered on
27 ml of 5-20% (wt/vol) sucrose-gradient made in the translation
buffer (5OmM Tris - HCI pH7.8/3.75mM magnesium
acetate/60mM NH4Cl), and centrifuged in Beckman SW-25.1
rotor at 35,00Oxg for 13 hrs at 0°C. Fractions were collected
from the bottom of the tube and the alkali-stable and TCA-insoluble radioactivity in each fraction was measured.
mRNA(UAA)
pppgeoeaeuug
ggouc
mRNA(UAG)
pppggogaawg
gguC
AGGGO OUCAG
mRNA(UGA)
pppggogaauug
gugoau
AGGAG GUCAG
See
AGGAGO
RESULTS
Design of synthetic messenger RNAs
The synthetic mRNAs used in this study are shown in Fig. 1.
A pair of codons to be tested, UAA, UAG or UGA, was preceded
by ten AUU isoleucine codons and followed by two UAA
termination codons.
1.5
0. 1.0
UAA
UG+Trp
0.5
-mRNA
0
2.5 5
E
A:
4.0
mRNA(UAA)
*.0
0.3
-b2.0
b22.0
vt 1.0
U
708
X t.0
50830
.
O~~~~~~
3.04 Jofi
10
4.0
20
__
30
40
708
-OH
E 4.0
03.0
ougcg
gaacuugausuc
-b2.0
PAt I
X1
C
10
ou
-OH
gou
mtin dill
4.0
b2
808 308
0
gs"cuugeueuo
uc
IO
An
0
40
An
Fraction number
D: mRNA(UGA)-Trp
2.
708
808 308
.0
S.c04
20
AUG AUU AUU AUU AUU AUU AUU AUU AUU AUU AUU UGA UGA UAA UAA
M
I
I
I
I
I
I
I
I
I
I *' VW*l
gaacuugauauc "gcu-OH
$a
u
308
.t
E
04
.04
ou04a
508
1.0
1.0
cUUC8
708
2.
3,1 04
s0
:
3.
B: mRNA(UAG)
Fraction number
C: mRNA(UGA)+Trp
X1g2.0
AUG AUU AUU AUU AUU AUU AUU AUU AUU AUU AUU UAG UAG UAA UAA
15
1.0
CL 3.0l
a
CL 3.0
AUG AUU AUU AUU AUU AUU AUU AUU AUU AUU AUU UM UAA UAA UAA
10
min.
Figure 2. Incorporation of [3H]isoleucine into alkali-stable and TCA-insoluble
materials in a cell-free translation of synthetic mRNAs. The codon to be tested
(test codon), UAA, UAG or UGA, in the mRNA is shown by triplet. '+Trp'
or '-Trp' indicates the translation in the presence or absence of tryptophan.
-mRNA: mRNA was not added.
GUCAG
SD.
UAG
UGA-Tr
20
40
0
80
Fraction number
FraCtion number
EE: +puromycin
Xl.0
708
808 308
20
5
30 40
Fraction number
3.04
Figure 1. Sequence alignment of three mRNAs, mRNA(UAA), mRNA(UAG)
and mRNA(UGA). The synthetic mRNAs include, 5' to 3' end, the
Shine-Dalgamo sequences (indicated as S.D.), an initiation methionine codon
AUG (M), ten AUU isoleucine codons (I), two test codons and two UAA
termination codons (*). Codons to be tested are UAA (*), UAG (*) and UGA
(W). Sequences shown by large letters are transcripts from insert DNAs; those
by small letters are from plasmid vector sequences. Regions from Sac I to Pst
I were used as the restriction enzyme sites for cloning. The Hind Em site was
used for linearization of plasmid and used as template.
10
Figure 3. Sucrose-gradient centrifugation of reaction mixture labelled with
[3H]isoleucie. (A) Translation of mRNA(UAA). (B) Translation of mRNA(UAG). (C) Translation of mRNA(UGA) in the presence of tryptophan. (D) The same
as (C) but in the absence of tsyptophan. (E) Translation of mRNA(UGA) followed
by incubation with puromycin.
Nucleic Acids Research, 1993, Vol. 21, No. 6 1337
Cell-free translation
Translation of the synthetic mRNAs was measured by
[3H]isoleucine as the labelled amino acid in the presence or
absence of tryptophan. With all mRNAs [mRNA(UAA),
mRNA(UAG) and mRNA(UGA)], [3H]isoleucine was
incorporated into TCA-insoluble materials with nearly equal
efficiency (Fig. 2). No incorporation occurred in the absence of
mRNA.
Sucrose-gradient centrifugation of reaction mixture labelled
with [3H]isoleucine
To investigate the state of the [3H]isoleucine-labelled peptides,
the incubated reaction mixture was examined by sucrose-gradient
centrifugation. In the case of mRNA(UAA) or mRNA(UAG),
the bulk of the synthesized peptide was recovered in the solublefraction (top of the gradient; Figs. 3A and B), indicating that
the peptides synthesized were released from the ribosomes at
UAA or UAG after translation of AUU isoleucine codons (Figs.
1 and 4A). With mRNA(UGA) in the presence of tryptophan,
the peptides were released from the ribosomes (Fig. 3C),
indicating that codon UGA was translated as tryptophan and the
peptide-chain termination occurred at UAA termination codon
following UGA (Figs. 1 and 4B). Some [3H]radioactivities in
the ribosome region in Figs. 3A, B, C would most probably
represent the unfmished [3H]isoleucine peptides in the course of
translation. When tryptophan was absent, the bulk of the peptides
was detected on the 70S ribosome fraction with some in the
soluble-fraction (Fig. 3D), indicating that the translation mostly
ceased at UGA without an appreciable release of the peptides
(Figs. 1 and 4C). Puromycin released the [3H]isoleucinelabelled peptides from the ribosomes completely (Fig. 3E).
Analyses with heterogeneous cell-free translation system
Since RF-2 of Escherichia coli or Bacillus subtilis recognizes
tenmination codons UAA and UGA (1, 3), the E. coli or B.subtilis
S-100 fraction was added to the M. capricolum S-30 fraction in
the presence of mRNA(UGA) and in the absence of tryptophan.
[3H]isoleucine was incorporated into alkali-stable and TCA-insoluble materials (Fig. 5), which were almost exclusively
recovered in the soluble-fraction after sucrose-gradient
centrifugation (Figs. 6A and B). No [3H]isoleucine
incorporation occurred in the absence of the M. capricolum S-30
fraction and in the presence of the E.coli or B.subtilis S-100
fraction (Fig. 5), indicating the absence of the ribosomes in the
S-100 fraction added.
min.
B:
mRNA(UGA) +Trp
CtI-
Figure 5. Incorporation of [3H]isoleucine into aLkali-stable and TCA-insoluble
materials with mRNA(UGA) in the presence of the E.coli and B.subtilis S-100
fraction and in the absence of tryptophan. Translation of mRNA(UGA) in the
presence of the M.capricol4m S-30 fraction and the E.coli (A) or B.subtilis (])
S-100 fraction. Translation with the M.capricolum S-30 fraction only (0).
Translation in the absence of the M. capricolum S-30 fraction but in the presence
of the Ecoli and B.subtilis S-100 fraction (0).
E
4.0
A: +E. collS-100
.O
B: + B. subtills S-1 00
C 3,!.0
3.0
n62.0
708
C
:mRNA(UGA)
-Trp
508
308
708
X 1 .0
508
308
04
0
<0
;l.
04
Ow
20
.
Fraction number
1
F0Fraction
t0 number
20
40
C
Figure 4. Models for cell-free translation of synthetic mRNAs. (A) Translation
of mRNA(UAA). The model can be applied to mRNA(UAG). (B) Translation
of mRNA(UGA) in the presence of tryptophan. (C) The same as (B) but in the
absence of typtophan. Figures (A) through (C) begin with the 10th AUU isoleucine
codon on the P site of the ribosome, so that the first test codon is on the A site.
RF in (A) and (B) (presumably RF-1) responds only to UAA and UAG. In (C),
in the absence of tryptophan, no further reaction occurs because of the absence
of the tryptophanyl-tRNA and RF (presumably RF-2) for UGA.
6. Sucrose-gradient centrifugation of reaction mixture labelled with
[3H]isoleucine in the presence of the E.coli or B.subtilis S-100 fraction. (A)
Translation of mRNA(UGA) with the Ecoli S-100 fraction. (B) The same as
(A) but with the B.subtlis S-100 fraction. (C) Model for translation. The figure
begins with the 10th AUU isoleucine codon on the P site of the ribosome, and
the first UGA codon on the A site. RF (presumably RF-2) in the E.coli and
B.subtilis S-100 fraction responds to UGA.
Figure
1338 Nucleic Acids Research, 1993, Vol. 21, No. 6
DISCUSSION
In Mycoplasma capricolum only UAA and UAG are used as
termination codons, and the unversal tenrination codon UGA
codes for tryptophan. The usage of UAA as a termination codon
is much higher than that of UAG because of high AT-pressure
(5). Oba et al. (8) showed that UAA acts as a termination codon
in the translation system of M.capricolum, resulting in release
of the synthesized peptides from the ribosomes. The experiments
described in this paper have shown that UAG also acts as a
termination codon, indicating that the RF recognizes both UAA
and UAG, and thus M.capricolum, like other bacteria, would
possess RF-1.
In Acholeplasma laidlawii, a close relative of M.capricolum,
UGA is not a tryptophan codon and is probably a termination
codon like in other eubateria (11). This suggests that the ancestor
had used three universal termination codons and therefore both
RF-1 and RF-2 would have existed. The appearance of UGA
as a tryptophan codon by several neutral changes in evolution
has been explained as follows (12). AT-pressure led to the
replacement of all UGA termination codons by UAA without
lehl effect. In AT-rich bacteria, the use of UGA as a termination
codon is rare, and most termination codons are UAA even in
E.coli (genomic GC content: 50%) (13). Therefore, almost
complete conversion of UGA to UAA could take place in the
Mycoplasma lineage. Appearance of UGA as a tryptophan codon
in the reading frames from mutation of UGG would require two
events before its appearance after separation from the
Acholeplasma lineage: (i) disappearance of UGA-recognizing
ability of RF so that UGA had become an unassigned codon,
and (ii) later emergence of tRNATA recognizing UGA as a
tryptophan codon. The latter event would have occurred by
recognizig the regular
duplication of the gene for
tryptophan codon UGG, and the anticodon of one of the
duplicates, under AT-pressure, mutated to UCA. The tandem
arrangement of the gene for tRNATrA and tRNACcA on the
chromosome of M. capricolum strongly supports this (6). If the
first event, i.e., production of unassigned codon UGA as an
intermediate step of this code change really took place, it is
expected that M.capricolum does not possess the UGA-dependent
RF-activity in response to UGA typtophan codon in the reading
frames. If the RE responds well to UGA, tRNAUCA acts only
as a suppressor tRNA, resulting in production of incomplete
peptides, with occasional readthrough of in-frame UGA codons.
The facts that the use of UGA tryptophan codon predominates
much over UGG (because of AT-pressure), and UGA was
translatd as trypphan as efficiendy as UGG in vitro (7) suggest
the absence of the RF-2 activity in this bacterium. However, there
has been no information on this. In this study, we have shown
that with mRNA(UGA) [3H]isoleucine-peptides were released
from the ribosomes in the presence of tryptophan; in its absence,
the bulk of the peptides was attached to the ribosomes. These
results suggest that the peptide synthesis ceased at the UGA site
and RF hardly responded to UGA. Some 'released' peptides in
the absence of tryptophan would most probably have resulted
from a small amount of tryptophanyl-tRNA that could not be
removed from the S-30 fraction by dialysis. These results indicate
that (i) UGA was translated as tryptophan and UAA following
UGA codons acted as a termination codon and that (ii) the UGAdependent RE-activity appears to be absent in the M. capricolum
S-30 fraction or, if it exists, its activity is much weaker an the
UAA- and UAG-dependent RF-activities.
It has been known that under amino acid starvation, uncharged
tRNA&P
tRNA occasionally pairs with a codon at the A site of the
ribosome, leading to the production of guanosine tetraphosphate
(ppGpp) (14). It is thus possible that in the absence of tryptophan,
uncharged tRNA pairs with UGA at the A site, so that UGAdependent RF, even if it exits, does not work. However, the
peptides attached to the ribosomes were completely released by
puromycin. This shows that the peptides are attached to the P
site and the A site is almost empty, i.e., not occupied by
uncharged tRNATFP, so that puromycin enters the A site and
therefore the RF for UGA, if present, could also enter there.
An addition of the Escherichia coli or Bacillus subtilis S-100
fraction to the M.capricolum S-30 fraction in the absence of
tryptophan, the synthesized peptides were completely released
from the ribosomes. E.coli and B.subtilis used are both tr
strains and tryptophan was exhaustively starved before
preparation of the S-100 fraction which were further dialyzed
much more exhaustively than the M.capricolum S-30 fraction.
The release of the synthesized peptides from M.capricolum
ribosomes by addition of these S-100 fractions almost certainly
resulted from recognition of codon UGA by RF-2 in the S-100
fraction of E.coli or B.subilis. Altogether, series of these results
support the hypothesis that UGAdepen4ent RF (presumably
RF-2) activity has disappeared so that 140A had become an
unassigned codon after separation of A4coplasma spp. and
Acholeplasma spp. from the common an¢tor.
Two possibilities exist to account for the present findings. (i)
RF-2 in M. capricolum is UAA-specific, or (ii) it is deleted from
M. capricolum. In rat mitochondria, UGA is a tryptophan codon
like in M.capricolum (15), and only one bacterial type RF-I that
recognizes UAA and UAG was deced (3). We therefore
consider that the second possibility is more plausible.
ACKNOWLEDGMENT
This work was supported by a grant from the Ministry of
Education, Science and Culture, Japan.
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