(Received for publication, March 27, 1995; and in revised form, May 25, 1995)
From the
Selenocysteine synthesis is achieved on a specific tRNA,
tRNA
Selenocysteine is a cysteine residue in which the thiol group is
replaced by the selenol group, SeH. This amino acid has been found in
prokaryotic and animal proteins that are generally involved in
oxidation-reduction reactions. Selenocysteine is not present in the
pool of natural amino acids. Rather, its cotranslational insertion into
polypeptide chains responds to a complex biochemical machinery, the
mechanism of which has been entirely unraveled in bacteria (see (1) and references therein for review). A specialized
selenocysteine, tRNA
Fig. 1A shows the 9-bp aminoacyl acceptor
stem of tRNA
Figure 1:
Phylogeny of the acceptor stem in
eukaryotic selenocysteine tRNAs and mutant constructs used in this
study. A, the vertebrate (Xenopus and mammalian)
acceptor stem is shown. Covariations occur in Drosophila at
5-68, 5a.67b, and 5b.67a. A C72 to U72 (in bold)
transition is found in Caenorhabditis elegans. The secondary
structure is from (5) , and the sequences are from (9) . B, G5a.U67b was replaced by either of the two
base pairs mentioned on the left, and U6.U67 was replaced by
one of the six base pairs on the right. C, base
substitutions at both G5a.U67b and U6.U67, which yielded either
G5a-C67b,U6.G67 or G5a-C67b,C6-G67, are indicated. D,
identities of the deleted base pairs mentioned in the text, indicated
by
Figure 2:
tRNA
Figure 3:
Kinetics of serylation of mutant
tRNA
Collectively, our data establish
that shortening the tRNA The issues underlying the work presented here originate from
inspection of the tRNA We elected to disclose
the function that could be born by the G5a.U67b and/or U6.U67 base
pairs by introducing a series of single or double point mutations at
these positions. The first step in selenocysteine synthesis is
represented by the charging of tRNA Two groups of
investigators established that the long extra arm and the discriminator
base G73 of tRNA When it comes to selenocysteylation, the
first prominent conclusion of our study resides in the fact that
tRNA Selenocysteylation of the tRNA In
prokaryotes, the tRNA The work presented here provides a step toward understanding the
mechanisms of eukaryotic serine to selenocysteine conversion. Other
issues await answers, for example the purification and cDNA cloning of
the eukaryotic SELB counterpart, whose existence was
evoked(22, 23, 24) , to decipher the identity
of its binding determinants on tRNA
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, which is first charged with serine to yield
seryl-tRNA
. Eukaryotic tRNA
exhibits an
aminoacyl acceptor stem with a unique length of 9 base pairs. Within
this stem, two base pairs, G5a.U67b and U6.U67, drew our attention,
whose non-Watson-Crick status is maintained in the course of evolution
either through U6.U67 base conservation or base covariation at
G5a.U67b. Single or double point mutations were performed, which
modified the identity of either or both of the base pairs. Serylation
by seryl-tRNA synthetase was unaffected by substitutions at either
G5a.U67b or U6.U67. Instead, and quite surprisingly, changing G5a.U67b
and U6.U67 to G5a-C67b/U6.G67 or G5a-C67b/C6-G67 gave rise to a
tRNA
mutant exhibiting a gain of function in serylation.
This finding sheds light on the negative influence born by a few base
pairs in the acceptor stem of tRNA
on its serylation
abilities. The tRNA
capacities to support
selenocysteylation were next examined with regard to a possible role
played by the two non-Watson-Crick base pairs and the unique length of
the acceptor stem. It first emerges from our study that tRNA
transcribed in vitro is able to support
selenocysteylation. Second, none of the point mutations engineered at
G5a.U67b and/or U6.U67 significantly modified the selenocysteylation
level. In contrast, reduction of the acceptor stem length to 8 base
pairs led tRNA
to lose its ability to efficiently support
selenocysteylation. Thus, our study provides strong evidence that the
length of the acceptor stem is of prime importance for the serine to
selenocysteine conversion step.
(
), is first
charged with serine by the conventional seryl-tRNA synthetase. The
product seryl-tRNA
is subsequently bound by
selenocysteine synthase, an enzyme which converts the seryl residue to
selenocysteine, using an activated phosphoselenoate compound as the
selenium donor. This activated compound is itself the product of the
SELD enzyme. Selenocysteine tRNA
is then brought to an in
frame selenocysteine-specifying UGA codon by SELB, a specific
elongation factor different from EF-Tu. Much less is known as to how
the eukaryotic machinery fulfills its role. The only identified protein
component consists in a fraction containing SELD and selenocysteine
synthase-like activities isolated from mouse liver(2) .
Eukaryotic selenocysteine tRNAs have been shown for quite some time to
recognize the UGA codon and to support serine to selenocysteine
conversion(3, 4) . In a previous work(5) , we
proposed a secondary and tertiary structure model for vertebrate
tRNA
. Its secondary structure deviates from that of a
classical elongator tRNA by the occurrence, among other structural
features, of exceptionally long aminoacyl acceptor and dihydrouracil
stems comprising 9 and 6 bp, respectively. On the basis of these
unusual characteristics, we asked whether base pairs in, or the unique
length of, the acceptor stem, with regard to 7 bp found in the majority
of elongator tRNAs, could reflect a function in selenocysteylation. In
this work, we first establish that tRNA
molecules
transcribed in vitro by T7 RNA polymerase can support
selenocysteylation in an in vitro assay. Second, we conclude
that the length of the acceptor stem constitutes one structural element
of tRNA
required for the serine to selenocysteine
conversion step.
tRNA Constructs
Synthetic bovine
wild-type and mutant selenocysteine tRNAs (6) were constructed
by hybridizing six couples of 14-24-mer oligodeoxynucleotides
containing the desired sequences. The sequences of the oligo couples,
which were used to synthesize the wild-type tRNA, are
given in Table 1. Mutants were constructed by swapping
appropriately one or several couples with oligos containing the
substituted sequences. Also included were the promoter of the T7 RNA
polymerase immediately 5` to the coding sequence and a BstNI
site CCAGG 3` to the coding region to generate the tRNA with a CCA
3`-end after linearization and transcription of the DNA templates. BamHI and EcoRI cloning sites located 5` and 3` to
the coding region, respectively, were also incorporated in the oligos.
After phosphorylation, the 12 oligos were combined, and the mixture
incubated at 90 °C for 10 min and then slowly cooled down to 25
°C. Intermolecular ligation of the oligos and to BamHI/EcoRI-cut pUC119 vector was performed overnight
at 16 °C.
In Vitro Transcription by T7 RNA
Polymerase
T7 RNA polymerase was prepared from the
overexpressing strain Escherichia coli BL21pAR1219 kindly
provided by F. William Studier. The purification protocol employed was
described in (7) . Conditions for transcription in vitro were as in (5) . The RNA products were purified by gel
electrophoresis and electroeluted. When necessary, 10% analytical gels
were run and stained with Stains-all for estimation of the ratio of
tRNA transcripts carrying an intact CCA end.
Serylation of tRNA
Prior to use,
tRNA transcripts were renatured by heating to 65 °C for 3 min and
then at 25 °C for 5 min. Aminoacylation occurred in 100 µl of
buffer containing 200 mM Tris-HCl, pH 7.4, 20 mM MgCl, 20 mM KCl, 10 mM ATP, 40
µM [
H]serine (29 Ci/mmol). For
plateau value determinations, tRNAs were added at a concentration of 3
µM, and the aminoacylation medium was incubated for
5-90 min at 37 °C. Kinetic parameters were determined from
three to six independent experiments with tRNA concentrations ranging
from 3 to 12 µM. Reactions were started by adding 4 µg
of a protein fraction containing partially purified bovine seryl-tRNA
synthetase prepared as described in (8) . 20-µl aliquots
were transferred onto pieces of Whatman 3 MM paper and submitted to 5%
trichloroacetic acid washes. Radioactivity remaining on the filters was
measured by scintillation counting.
Selenocysteylation
HSe
was prepared enzymatically from radioactive sodium selenite
(DuPont NEN) and chromatographically purified according to (2) . In vitro synthesis of
[
Se]selenocysteyl-tRNA
was
performed as described in (2) . Briefly, tRNA
(25-100 ng) was incubated for 2 h at 30 °C in a
50-µl reaction mixture comprising 200 mM Hepes-NaOH, pH
7.0, 20 mM MgCl
, 20 mM KCl, 5 mM ATP, 0.4 mM serine, 5 mM 2-mercaptoethanol, 0.7
µM H
Se
(1-2 µCi),
3 µg of a fraction containing partially purified seryl-tRNA
synthetase, and 10 µl of a fraction containing the
selenide-activating and selenocysteine synthase activities. After
ethanol precipitation, alkaline hydrolysis of the
[
Se]selenocysteyl-tRNA
released
[
Se]selenocysteine, which was separated by TLC
on silicagel G plates in n-butanol:acetic acid:water (4:1:1).
Se radioactivity was measured with a Fuji BioImage BAS
2000 analyzer. Cold selenocysteine (a generous gift of Prof. K. Soda,
Kyoto University) was cochromatographed as a control and revealed by
ninhydrin reaction.
and the sequence covariations, which allow
its maintenance in the course of evolution(5, 9) .
When comparison is restricted to the non-canonical (non-Watson-Crick)
base pairs G5a.U67b and U6.U67, one can observe that the latter is
strictly conserved in identity. The non-canonical status at position
5a.67b is maintained with, however, a G5a.U67b to A5a.G67b covariation,
which was allowed in Drosophila. Conservation of or
covariations at non-Watson-Crick base pairs aiming at maintaining a
non-canonical status in RNA helices are often indicative of an
important structural or functional role played by these particular base
pairs(10, 11, 12, 13, 14) .
In connection with this and the occurrence of an unusually long
acceptor stem, we asked two questions: 1) is there a function devoted
to these non-Watson-Crick base pairs and 2) what role can be attributed
to the long acceptor stem?
.
A 2-Base Pair Substitution in the Acceptor Stem Leads to an
Improved Serylation Mutant
The first step in the
selenocysteine insertion machinery consists in the charging of
tRNA with serine. Thus, to determine if the
aforementioned non-Watson-Crick base pairs could exert a function, we
first wished to know whether altering this base pairing scheme would
affect serylation of tRNA
. To this end, a series of
single or double point mutations was performed, which substituted
G5a.U67b and U6.U67 (Fig. 1B). All four possible
Watson-Crick pairs and G.U or U.G pairs were engineered by introducing
four single and two double point mutations at the U6.U67 base pair.
Separately, G5a.U67b was substituted to a Watson-Crick G-C pair and to
the non-canonical A.G pair found in Drosophila (Fig. 1B). The tRNA
variants
carrying these mutations were produced by in vitro transcription with T7 RNA polymerase. The calculated relative V
/K
values (Table 2) for the mutants compared to wild-type tRNA
are in the range of 0.5-3, reflecting an almost negligible
effect of the substitutions. We therefore concluded that the
replacements made separately at G5a.U67b or U6.U67 did not influence
significantly serylation of tRNA
. We next produced
substitution mutants of both the G5a.U67b and U6.U67 base pairs, giving
rise to two different tRNA
variants carrying the combined
substitutions G5a-C67b/U6.G67 or G5a-C67b/C6-G67 (Fig. 1C). The V
/K
ratio of
G5a-C67b/U6.G67 increased by a factor 6 compared to wild-type
tRNA
(Table 2). This value results from a decrease
in the apparent K
(8.5 µM)
and an increase in V
(475 units). The surprise,
however, arose from mutant G5a-C67b/C6-G67, which exhibited a dramatic
increase in the V
/K
ratio, becoming 17.5 times as high as that of wild-type
tRNA
(Table 2). Both a significant drop in the
apparent K
value (5 µM) and
an increase in the V
(875 arbitrary units)
contribute to the obtention of this unexpected finding. Thus, point
mutations changing G5a.U67b to G5a-C67b or U6.U67 to either U6.G67 or
C6-G67 did not provoke significant effects. In marked contrast, the
combined substitutions G5a-C67b/U6.G67 and G5a-C67b/C6-G67, and more
prominently the latter one generating an almost all G-C pair acceptor
stem, endows serylation of these tRNA
variants with a
higher V
/K
ratio
than the wild type.
Selenocysteylation Is Tolerant to Base Pair
Replacements in the Acceptor Stem
Utilization of an in
vitro assay for serine to selenocysteine conversion was primarily
reported in (2) and is described under ``Experimental
Procedures.'' As the assay was originally established for native
tRNA purified from bovine liver, a prerequisite was to
verify that an in vitro transcribed tRNA
(T7tRNA
) was also able to support
selenocysteylation. Fig. 2A shows this is indeed the
case since 25 ng of native tRNA
or T7tRNA
(lanes1 and 2, respectively) gave
rise to [
Se]selenocysteine spots of similar
intensity. When the number of femtomoles of
[
Se]selenocysteine produced in the assay by
T7tRNA
was plotted versus the T7tRNA
concentration, the dose-product relationship reached a plateau at
10 nM of tRNA
(Fig. 2B). Thus,
in further experiments, 25 or 100 ng of T7tRNA
,
corresponding to approximately 15 and 55 nM in the assay, will
be used. All the substitution mutants that changed singly G5a.U67b or
U6.U67, or those that substituted both base pairs together (see Fig. 1, B and C), were assayed for their
capacities to be selenocysteylated. Table 2reports the
percentage of selenocysteylation of the tRNA
variants
with respect to the in vitro transcribed wild-type
tRNA
. It ranges from 75% for the U6.U67 to U6.G67 and
C6-G67 covariations to 98% for the G5a.U67b to G5a-C67b base
replacement. This indicates that values fluctuate moderately and do not
deviate significantly from that provided by wild-type
tRNA
. From these results, we conclude that base
replacements or covariations engineered at some targeted base pairs in
the amino acceptor arm do not affect selenocysteylation under our
conditions.
transcribed in
vitro by T7 RNA polymerase can support efficient
selenocysteylation. A, autoradiograph showing selenocysteine
produced from tRNA
transcribed in vitro (lane1) or native tRNA
(lane2). Position of selenocysteine and the origin of the
chromatograph are indicated by Sec and O,
respectively. B, curve representing the amount of
selenocysteine produced versus tRNA
concentration under limiting enzyme.
The Length of the Amino Acceptor Arm Is Important for
Selenocysteylation
To answer the second question aiming at
assigning a specific role to the particularly long acceptor stem, base
pair G5a.U67b or U6.U67 was deleted, giving rise to mutant
(G5a.U67b) or
(U6.U67), represented in Fig. 1D. Aminoacylation kinetics were performed, which
indicated that the tRNA
deletion mutants possess a
slightly better capacity of charging serine than the wild type (Fig. 3). The variability observed in the plateau values is not
caused by differences in the tRNA quantities but is rather due to the
well known plateau effects, which take into account the spontaneous and
enzymatic deacylation of tRNAs(15) . Thus, 100% of charging
cannot be attained, and what was observed very likely reflects the
effects of the mutations. These deletion mutants were next assayed for
their abilities to support serine to selenocysteine conversion.
Surprisingly, Table 2shows that both deletions induced a marked
down effect on selenocysteine synthesis. However, the intensity of
inhibition is not identical in both cases.
(U6.U67) still enables
the tRNA
carrying this deletion to support
selenocysteylation to 24% of the wild-type level, while
(G5a.U67b)
gives rise to only 12% of selenocysteylation, a value that we
considered as strongly deleterious. To determine whether this was due
to deletion of these particular base pairs or to a more general effect
caused by reduction of the stem length to 8 bp, two other deletions,
(C3-G70) and
(A5b.U67a), were generated, which removed C3-G70
and A5b.U67a, respectively (Fig. 1D). These base pairs
are situated at two different locations in the stem. Both deletions
conferred mitigating up effects on the serylation capacities of the
tRNA
variants (Fig. 3). Evaluation of the
selenocysteylation abilities of these mutants revealed that both
(C3-G70) and
(A5b.U67a) achieved a strong repression, leaving
10 and 14% of residual selenocysteylation, respectively (Table 2). Thus, similarly to
(G5a.U67b), these deletions
led to tRNA
variants unable to convert serine to
selenocysteine. Incubation of 100 ng, instead of 25 ng, of the four
tRNA
deletion mutants in the assay did not lead to a
proportional increase in selenocysteine synthesis. Indeed, it emerges
from Table 2that
(C3-G70),
(G5a.U67b) and
(A5b.U67a), which disabled tRNA
when 25 ng were
used, marginally improved the selenocysteylation abilities since the
deletion mutants allow about 25% of selenocysteine to be made.
Likewise, 37% of selenocysteine was obtained with the
(U6.U67)
mutant under the same conditions. This indicates that the wild-type
selenocysteylation abilities abrogated by the base pair deletions could
not be significantly restored by augmenting the tRNA
amount in the assay, thus corroborating the observations made
with 25 ng of tRNA
.
carrying deletions in the acceptor stem. Plateau
values of wild-type and mutant derivatives carrying the base pair
deletions are shown in Fig. 1D.
amino acceptor stem to 8 bp is
severely detrimental to selenocysteylation in vitro. It
appears, however, that removal of U6.U67 leads to a less penalizing
effect than deletion of the C3-G70, G5a.U67b, or A5b.U67a base pair.
secondary structure and sequence
comparisons(5, 9) . In the first place, the occurrence
of a 9-bp amino acceptor stem, instead of 7 bp in the vast majority of
the eukaryotic elongator tRNAs, constituted the central question that
guided our investigations. Within this long stem, two non-Watson-Crick
base oppositions, G5a.U67b and U6.U67, were shown in our previous work
to actually pair in solution(5) . Interestingly, we observed
that the non-Watson-Crick (non-canonical) status of base pair U6.U67 is
evolutionarily maintained by a strict conservation of both U6 and U67,
while the non-canonical status at position 5a.67b subsists during
evolution as a result of either sequence conservation or G5a.U67b to
A5a.G67b covariation (Fig. 1A). Occurrence of
non-Watson-Crick base pairs, their conservation or maintenance by
compensatory changes, has been listed in a variety of RNAs such as
tRNAs(10, 11) , 5 S RNAs(12) , ribosomal
RNAs(13) , and the hammerhead ribozyme(14) , reflecting
important structural and/or functional roles.
with serine. It was
therefore logical to assess beforehand the possible repercussions of
the mutations on the serylation of tRNA
. None of the
single base or base pair replacements carried out separately at
G5a.U67b or U6.U67 affected in a significant manner serylation of the
tRNA
mutants. Much to our surprise, however, the double
base pair substitutions G5a-C67b/U6.G67 or G5a-C67b/C6-G67 promoted a
strong augmentation of the V
/K
ratio. This
constituted an unpredictable finding from inspection of the
tRNA
structure. These results underline the notion that
tRNA
is not optimized for the serylation step. Likewise,
it was reported that prokaryotic tRNA
is not an excellent
substrate for prokaryotic seryl-tRNA synthetase since the K
/K
value is only
1% that of tRNA
(16) .
function as major identity elements for
seryl-tRNA synthetase(17, 18) . Our finding proposes
that some base pairs in the acceptor stem can negatively influence
serylation of tRNA
. In the absence of a structural model
describing in detail the interaction between tRNA
and
seryl-tRNA synthetase, it is perilous to interpret our data. However,
it may well be that the seryl-tRNA synthetase establishes base-specific
and/or backbone contacts at the acceptor stem of tRNA
(serine). These would be disadvantaged by the sequence of some
bases in the wild-type tRNA
, explaining the revival of
activity observed with the two tRNA
mutants. This type of
contact was reported to contribute, among others, to the interaction
between prokaryotic tRNA
and seryl-tRNA
synthetase(19) .
transcribed in vitro by T7 RNA polymerase
can efficiently support selenocysteylation in a similar fashion to
native tRNA
. This suggests that the structure of the
tRNA
perse is sufficient to govern
recognition by selenocysteine synthase, the lack of modified bases in
the tRNA
transcript being apparently not redhibitory to
selenocysteylation.
variants harboring base changes at G5a.U67b and/or U6.U67
revealed that none of the mutants affected the level obtained with the
wild-type tRNA
transcript. In marked contrast,
exploitable information was furnished by reduction to 8 bp of the
length of the acceptor stem. All four deletions,
(C3-G70),
(G5a.U67b),
(A5b-U67a), and
(U6.U67), impede the
selenocysteylation function of the tRNA
mutants carrying
these deletions. A certain disparity was nevertheless observed in the
impediment level since the first three mutants obliterate the activity
while
(U6.U67) reduces dramatically selenocysteylation but does
not abolish it. This was interpreted to mean that the length of the
acceptor stem of tRNA
plays an important role in the
serine to selenocysteine conversion step. Conceivably, the residual
level obtained with
(U6.U67) could reflect a position-dependent
effect of the deletions. One hypothesis would be that selenocysteine
synthase not only recognizes the 9-bp acceptor stem but also
establishes essential base or backbone-specific contacts with part only
of this stem. The U6.U67 deletion would then be less detrimental than
others since it is located at the bottom of the stem (Fig. 1A). Alternative possibilities certainly exist,
but, as for the serylation mutants, the lack of a model displaying the
tRNA
-selenocysteine synthase interactions renders highly
speculative any structural interpretation. Despite this, it must be
stressed that our phenomenological description remains valid and does
highlight the prevalent requirement for a 9-bp acceptor stem.
acceptor stem is 8 bp
long(20, 21) , namely 1 base pair longer than
canonical tRNAs but 1 base pair shorter than its eukaryotic
counterpart. Shortening to 7 bp reduces selenocysteine-synthase
activity but does not compromise it, while this shortened stem prevents
recognition by SELB, thus enabling EF-Tu to bind
tRNA
(16) . This finding underscores a notable
difference between prokaryotes and eukaryotes for the
selenocysteine-synthase recognition elements on tRNA
.
. In particular, it
might well be that the non-canonical status of base pairs G5a.U67b and
U6.U67 is important for recognition of tRNA
by the
eukaryotic SELB factor.
,
selenocysteine tRNA; bp, base pair.
We are grateful to Catherine Florentz and Richard
Gieg for helpful discussions. Christine Loegler
is thanked for skillful technical assistance and Annie Hoeft for
oligonucleotide synthesis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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Copyright © 1995 by the American Society for Biochemistry and Molecular Biology.
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Molecular and Cellular Proteomics
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