1993 Oxford University Press
Nucleic Acids Research, 1993, Vol. 21, No. 24
5589-5594
The long extra arms of human tRNA(Ser)Sec and tRNASer
function as major identity elements for serylation in an
orientation-dependent, but not sequence-specific manner
Xin-Qi Wu and Hans J.Gross*
Institut fur Biochemie, Bayerische Julius-Maximilians-Universitat, Biozentrum, Am Hubland,
D-97074 Wurzburg, Germany
Received September 9, 1993; Revised and Accepted November 1, 1993
ABSTRACT
Selenocysteine tRNA [tRNA<Ser>Sec] is charged with
serine by the same seryl-tRNA synthetase (SerRS) as
the canonical serine tRNAs. Using site-directed
mutagenesis, we have introduced a series of mutations
into human tRNA<Ser>sec and tRNASer in order to study
the identity elements of tRNA<Ser)Sec for serylation and
the effect of the orientation of the extra arm. Our results
show that the long extra arm is one of the major identity
elements for both tRNASer and tRNA<Ser>Sec and gel
retardation assays reveal that it appears to be a
prerequisite for binding to the cognate synthetase. The
long extra arm functions in an orientation-dependent,
but not in a sequence-specific manner. The discriminator base G73 is another important identity element
of tRNA<Ser)Sec, whereas the T- and D-arms play a
minor role for the serylation efficiency.
INTRODUCTION
The recognition of tRNA by its cognate synthetase is one of the
crucial steps towards the fidelity of protein synthesis. The
anticodon bases, the discriminator base at position 73, sequences
in the acceptor stem and the variable pocket have been shown
to be the identity elements in a number of class I tRNAs which
contain an extra arm of 4 or 5 nucleotides (for review see refs.
1-3). The class H tRNAs, which include tRNA 5 ", tRNA^"
and prokaryotic tRNATy, are characterized by their long
variable extra arms of 10 or more nucleotides (4). The long extra
arm of Escherichia coli tRNASer has been shown to play a
critical role in discrimination against the other two class II tRNAs
by the cognate synthetases (5,6). Crystallographic structures of
E. coli seryl-tRNA synthetase complexed with tRNA 5 " have
revealed that the long a-helical arm of the synthetase is inserted
between the TYC-loop and the long variable arm, making
contacts with both (7). The protection of sequences in the long
variable arm from chemical modification in the yeast
tRNA^Vsynthetase complex also suggested the contact of this
arm with the synthetase (8). More recently, the long extra arm
has been shown to be one of the major identity elements of human
:
To whom correspondence should be addressed
tRNA&r by introducing it into tRNAVal (9). However, how
exactly the long extra arm functions as a major identity element
remained to be elucidated.
Selenocysteine tRNA which recognizes specific UGA stop
codons in some mRNAs and inserts selenocysteine residues into
the nascent polypeptides has been identified in both prokaryotes
and eukaryotes (10-13). tRNA(Ser)Sec is first serylated by the
same seryl-tRNA synthetase which charges the canonical serine
tRNAs (10,14). Seryl-tRNAt^ 5 " is then converted into
selenocysteyl-tRNA'5"*560 in the presence of selenocysteine
synthase and other proteins (15 — 17). The secondary structures
of E. coli and vertebrate tRNAs(Ser)Sec have recently been
deduced by structure probing (18,19). In contrast to the isoacceptors of other tRNA species, tRNAs(Ser)Sec from vertebrates
are quite different from the canonical tRNAs5" in their primary
structure, especially in the sequence and length of the variable
arm (4). Their secondary and tertiary structures are also different
from the common tRNAs 5 " in some respects (19,20). Thus, it
is of interest to disclose the features of tRNA(Ser)Sec which are
recognized by vertebrate seryl-tRNA synthetase.
In this work we show that the long extra arm and the
discriminator base G73 are the major identity elements for the
serylation of human tRNA(Ser)5ec The long extra arm is, in
an orientation-dependent, but not sequence-specific manner,
necessary for serylation of human tRNA(5er)5ec and tRNA5cr and
is a contact site for binding of the seryl-tRNA synthetase to its
cognate tRNA.
MATERIALS AND METHODS
Enzymes and reagents
T7 RNA polymerase was prepared in our laboratory from an
overproducing strain kindly provided by Dr. W. Studier (21).
Cytoplasmic Si00 extract from HeLa cells used as the source
of synthetases was prepared according to Dignam et al. (22).
3
H-Serine (1.33 TBq/mmol) and [a-32P]GTP (111 TBq/mmol)
were from Amersham. Other enzymes and reagents were
purchased from commercial suppliers.
5590 Nucleic Acids Research, 1993, Vol. 21, No. 24
Bacterial strains and plasmids
E. coli JM 109 was used as host for the propagation of plasmid
pUC19. E. coli strains CJ236 and TGI were used for site-directed
mutagenesis in M13 vectors. pHtU contains the 0.5 kb
EcoRI/Aval fragment coding for human tRNA(Ser)Sec (23) cloned
in pUC19. pHtS contains a synthetic tRNASer gene deduced
from the tRNA^CUGA) sequence (24). The tDNAs in plasmids
pHtU and pHtS are immediately preceded by a T7 promoter
sequence and are followed by the BstNI recognition site CCAGG
in order to generate a mature CCA 3'-end of the transcripts.
Site-directed mutagenesis
Oligonucleotide-directed mutagenesis was carried out in
ssM13mpl8 or M13mpl9 (25). Mutations were confirmed by
dideoxynucleotide chain termination DNA sequencing (26).
In vitro transcription of tRNA genes with T7 RNA polymerase
In vitro transcription with T7 RNA polymerase was performed
as described by Himeno et al. (27). tRNAs transcribed were
purified on a 10% polyacrylamide/8 M urea gel and eluted from
the gel slices with TE buffer (10 mM Tris-HCl, 1 mM EDTA,
pH 8.0) containing 10% phenol.
In vitro aminoacylation of tRNA
Aminoacylation of tRNA was performed at 37 °C in 20 jtl of
aminoacylation buffer (20 mM imidazole—HC1, pH 7.5; 150 mM
KC1, 8 mM MgCl2, 0.5 mM DTT), 5 mM ATP, 0.5 mM CTP,
5 jtM 3H-serine (0.17 TBq/mmol), 0.33 /xM tRNA and 15%
(v/v) of S100 extract (3.6 mg/ml) from HeLa cells. 3 ml aliquots
were transferred in 5 min intervals onto 1 cm2 pieces of
Whatman 3 MM paper and submitted to trichloroacetic acid (TCA) wash (15 min in 10% cold TCA followed by 3 x 5 min in
5% cold TCA). The radioactivity remaining on the filters was
measured by scintillation counting.
Preparation of 32P-labeled tRNA
32
P-Labeled tRNA was obtained by in vitro transcription with
T7 RNA polymerase as described above, except that GTP was
replaced by 0.2 mM [a-32P]GTP (0.18 TBq/mmol). The
transcription was scaled down to 1 /tg of template DNA.
Gel retardation assay for competition of tRNAs for synthetase
binding
2 ml of the SI00 extract (3.6 mg/ml) from HeLa cells were
incubated in aminoacylation buffer in a volume of 10 /il with
a total RNA preparation (10 fig) from E. coli for 20 min at 0°C,
then with unlabeled competitor tRNAs (0.3 - 0 . 8 /*M) for 20 min
at 0°C, followed by 32P-labeled tRNA** (10000 cpm, about
0.03 |tM) for further 10 min at 0°C. The tRNA/synthetase
complex was analysed on a 4% polyacrylamide gel
(acrylamide/bisacrylamide = 66:1) which was run in TB buffer
(0.09 M Tris-borate, pH 8.0) at 4°C.
RESULTS
Effect of mutations in the long extra arms of human
tRNA<Ser>Sec and t R N A ^ on serylation
Replacement of the long extra arm of tRNA*5"'5** and tRNA 5 "
by the short extra arm of tRNAVal as shown in cases of
tRNA(Ser)SecXi and t R N A ^ l (Fig. 1) abolished serylation
(Fig. 2A and 2B). The serylation of tRNA^Yl could be
partially restored by replacement of its short extra arm with the
long extra arm from tlUNA^'i^ as shown in tKNASerY3
(Figs 1 and 2B). The orientation of the extra arm in tRNA^YS
is [a(l)/3(2)], where a and /3 are the number of unpaired
nucleotides between the anticodon stem and the extra arm and
between the extra arm and the T-stem, respectively. This is like
that of tRNA5" [a(l)/3(2)]. tRNASerY2 (Fig. 1) has also an
extra arm from tRNA^ 5 **, but its orientation [a(2)/3(0)] is
different from that of the wild-type tRNA5" [a(l)/3(2)]. This
mutant was only very weakly serylated (Fig. 2B). However,
tRNASerY4 with an extra arm from tRNASer in an orientation
[a(2)j3(O)] similar to tRNASerY2 (Fig. 1) is significantly
serylated (Fig. 2B). Mutation of G47:A of tRNA&rY4 to A47:A
yielding Y5 (Fig. 1) dramatically reduces serylation (Fig. 2B).
The exchange of G26 in tRNASer to U26 did not affect
serylation (Fig. 2B, Y6).
Changing the orientation of the extra arm of tRNA(Ser)See
[a(2)/3(l)] to that of tRNA(Ser'SecX2 [a(l)/3(l)] by deletion of
G45 (Fig. 1) slightly impairs the charging activity (Fig. 2A). A
further C insertion between G47:L and A48 as shown in
tRNA(Ser'SecX3 [a(l)j8(2)] dramatically reduces serylation (Fig.
2A). An even lower charging efficiency is obtained for mutant
tRNA(Ser)SecX4 [0,(0)0(2)] (Fig. 2A), which has an additional
A44 deletion as compared with tRNA(Ser>SecX3 (Fig. 1).
Mutation of A44G45 in tRNA(Ser>Sec to U44C45 slightly reduces
serylation (Fig. 2A, X5).
Effect of mutations at the discriminator position and other
domains of tRNA<Ser'Sec on serylation
The change of the discriminator base G73 to A73 or C73
eliminates the serylation of t R N A ^ * * (Figs 1 and 2A, X6 and
X7). Replacement of the acceptor stem of tRNA<Ser>Sec by that
of tRNASer does not affect serylation (Figs 1 and 2A, X8).
Exchange of the acceptor stem and T-stem of tRNA*5")580 by
those of tRNA5" reduced serylation, but did not abolish it (Figs
1 and 2A, X9). Substitution of G22 to A22 in t R N A ^ ^ X l O
(Fig. 1) caused a major loss of aminoacylation (Fig. 2A). In
tRNA<Ser>5ecX10, there is an additional G insertion next to U26
(Fig. 1). However, this insertion alone does not significantly
affect serylation (not shown). Substitution of the whole D-stem
of t R N A ^ ' ^ by that of tRNASer as shown in tRNA(Ser>SecXl 1
abolished serylation (Figs 1 and 2A).
Effect of the extra arm on the binding of tRNA^ and
tRNAl 5 * 3 ^ to the seryl-tRNA synthetase
Gel retardation is often used to detect the specific interaction
between proteins and nucleic acids (29). Application of gel
retardation to study the specific binding between tRNA and its
cognate synthetase has been reported (30). As shown in Figure 3,
the 32P-tRNA/SerRS complex is formed upon incubation of
labeled human tRNA 5 " with S100 extract from HeLa cells
(Fig. 3, lane a). Complex formation is inhibited by preincubation
of the SI00 extract with unlabeled tRNA5" (Fig. 3, lane b), but
not by tRNAVal (Fig. 3, lanes k and 1). The contact of the long
extra arm of yeast tRNASer with yeast synthetase has been
suggested by the protection of the extra arm from chemical
modification in the presence of the enzyme (8). As mentioned
above, t R N A ^ ^ l and t R N A ^ ^ X l with a short extra arm
are not serylated (Fig. 2). Therefore, we conclude that the long
extra arm is a prerequisite for the binding of tRNA 5 " and
tRNA<Ser)Sec to the synthetase. To confirm this, a competition
Nucleic Acids Research, 1993, Vol. 21, No. 24 5591
JUOO U
U0»CUCCU
C O
™UCUOOOO
pppo-c
U-A
A-U
O-C
U-A
CO
,"°AoccooU
COUCCU
I I I
'"u**O«Ou
Figure 1. Secondary structures of human tRNA(Ser)Sec, tRNA 5 " and their derivatives (T7 RNA polymerase transcripts). Mutations are boxed and the empty boxes
([ ]) indicates nucleotide deletions. The secondary structure and numbering of tRNA(Ser)Sec are according to Sturchler et al. (19). The numbering of tRNA 5 " is
according to Sprinzl et al. (28).
assay was performed with tRNA<Ser)Sec and a number of mutants
tRNAs. While the subsequent binding of 32P-labeled tRNA5" to
the SerRS is impaired by the pre-incubation of the extract with
wild-type t R N A ^ 5 " (Fig. 3, lane g), or mutant tRNAs with
a long extra arm (Fig. 3, lanes e, f and h), as by pre-incubation
with the wild-type tRNA**, it is not inhibited by mutant
5592 Nucleic Acids Research, 1993, Vol. 21, No. 24
160&
1600
15
15
Time (min)
Time (min)
Figure 2. Kinetics of aminoacylation of human tRNA(Scr)Sec, tRNA^and their derivatives. (A) Serylation of tRNA(Ser)Sec and its derivatives XI to Xll (Figure 1)
by HeLa synthetase. (B) Serylation of tRNA 5 " and its derivatives Yl to Y6 (Figure 1) by HeLa synthetase.
or tRNA<Ser)Sec with a short extra arm (Fig. 3, lanes c
and j). tRNASerY2 and tRNA<Ser>SecX4, which are very weakly
serylated due to the change of the orientation of their extra arms
(see above), have a reduced (Y2) or weak (X4) ability for
competition with 32P-labeled tRNASer (Fig. 3, lanes d and i).
The competition strength of the competitor tRNAs with
tRNASer, in general, correlates well with their charging
efficiency with 3H-serine.
DISCUSSION
In the present work we used tRNA(Ser)Sec and tRNASer
synthesized in vitro by T7 RNA polymerase to characterize the
identity elements of human tRNA(Ser)Sec for serylation and the
effect of the orientation of the long extra arm on the serylation
of human tRNA(Ser>Sec and tRNA 5 ". tRNA^ 1 -)^ and tRNA 5 "
with a short extra arm from tRNAVal are not serylated (Fig. 2),
and like tRNAVal, cannot compete with the binding of tRNA 5 "
to the seryl-tRNA synthetase (Fig. 3). This implies that the long
extra arm is a prerequisite for binding of SerRS to its cognate
tRNA. In consistence with this conclusion, the extra arm of yeast
tRNASer was suggested to be in contact with the synthetase (8).
Structure probing with Pb2"1" revealed that the replacement of the
extra arm of tRNA &r by that of tRNAVal does not significantly
alter the overall three-dimensional structure as evidenced by the
specific cleavage patterns reflecting the interaction between the
T- and D-stem or -loops (data not shown). Nucleotide changes
that disrupt the tertiary interactions of tRNA"16 alter the
cleavage pattern even if they are distant from the Pb 2 * binding
pocket (31). Furthermore, the precursor of tRNA^Tl is
matured as efficiently as the wild-type by the processing enzymes
(data not shown), which are considered as sensitive indicators
for a correct three-dimensional structure (9,32). Thus, a dramatic
disorder of the three-dimensional structure caused by the
substitution of the extra arm seems to be unlikely. The binding
and serylation of tRNA 8 " with a short extra arm can be partially
restored by a long extra arm originating from tNRA*5")5'* (Figs
1, 2B and 3, Y3). It has been reported that E. coli and bovine
tRNA(S")5ec and tRNA 5 " are serylated by the same synthetase
from E. coli and bovine liver, respectively (10,14). The similar
serylation and competition behaviour between human
tRNACSe1^ ^ d tRNA 5 " and their mutants also suggest that
tRNA(Ser)Sec and tRNA 5 " are serylated by the same human
-
+
- (-) +
+
•
+ (+) (-)
-
-
-
•
•
•
-
t
c
t
u
•1
mil'
II
IIi
i. »
1
a
b c
d
e
f
g
h
i
j
k
Figure 3. tRNA/synthetase complex formation of 32P-labeled tRNASer in HeLa
S100 extract. Lane a, water; in the presence of competitors: lanes b-f, tRNA&r,
Yl, Y2, Y4 and Y3; lanes g - j , tRNA(Ser)Sec, X3, X4 and XI; lanes k - 1 ,
tRNAVal (0.4 and 0.8 pM). The concentration of the competitor tRNAs except
tRNAVal is 0.3 nM. The positions of 32P-tRNASer (t), an unknown complex (u)
and the tRNA/SerRS complex (c) are indicated at the right side. The 'u' bands
appear to be unspecific complexes since they do not disappear upon competitor
(tRNASer) addition (lane b). The charging activity of each competitor tRNA with
serine is indicated on the top by + , (+), ( - ) or - .
SerRS. Therefore we conclude that the long extra arm of
tRNA(Ser)5ec is one of the essential identity elements for the
serylation of human tRNA(S")Sec as has also been shown for the
serylation of human tRNASer by switching human tRNAVal into
a serine acceptor (9).
The long extra arm of tRNA** and tRNA(Ser)Sec does not
function in a squence-specific manner. There is only one base
pair identical in the extra arms of tRNASer (G46-C47:G) and
t R N A * ^ (G46-C47:H), yet tRNASerY3 is significantly
serylated (Fig. 1 and 2B). This G - C base pair is not present
in rat tRNA 5 " with anticodon GCU (which has Y46-A47:G)
and vertebrate tRNAst 5 "^ where there is U47-A47:K at the
same position (4). The last base pair in the extra arm is
G47:A-C47:E or C47:A-G47:E in all eukaroytic serine
tRNAs5" (4). However, mutation of G47:A to A47:A did not
significantly affect serylation (Breitschopf and Gross, unpublished). Moreover, E. coli tRNA 5 " and rat tRNA&r can be
charged with serine by yeast synthetase (33-35) although the
Nucleic Acids Research, 1993, Vol. 21, No. 24 5593
extra arm of E. coli tRNA 5 " is quite different from that of
eukaryotic tRNA5" (4). The sequences in the extra arm of
tRNAs(Ser)Sec from the animal kingdom have undergone some
evolutionary variations (36). A similar situation also exists for
E. coli tRNAs5". The extra arm of E. coli tRNA 5 " is also
required for recognition by SerRS (5, 6). However, the sequence
and the length of the extra arm within E. coli serine isoacceptors
including E. coli tRNA^s*
are quite different (4). It is
proposed that the positive effect of the extra arm on serine identity
is due to its structure rather than its sequence (6).
The orientation of the long extra arm can dramatically affect
serylation as revealed by the apparent difference in serylation
resulting from the difference in the orientation of the extra arm
between tRNAs<5">5ec [a(2)/3(l)], X3 [a(l)/3(2)] and X4
[a(0)|8(2)] (Figs 1 and 2A). An obvious improvement of
serylation was observed by changing the orientation of the extra
arm in tRNASerY2 to that in tRNASerY3 (Figs 1 and 2B). An
extreme example is E. coli tRNA1*', where a change of the
orientation of its extra arm by insertion of two nucleotides allows
it to be serylated (5). The effect of the orientation of the extra
arm may occur through affecting the association between the
tRNA and the synthetase as suggested by the reduced competiting
ability of tRNA(Ser)5ecX4, which has a change in the orientation
of the extra arm (Figs 1 and 3). The orientation may also affect
the interaction of the bound synthetase with other identity
elements. One such element is the discriminator base G73.
Mutation of G73 to A73 or C73 abolishes serylation of tRNA5"
(9) and tRNAt5")5"* (Fig. 2A, X6 and X7).
The orientation of the extra arm of tRNASer as well as
tRNA(Ser)Sec may not be determined merely by the number of
unpaired bases at the basis of the extra arm. The nature of the
sequences in the variable helix may also have some influence
on its orientation. This is shown in the cases of tRNASerY2 and
tRNASerY4. They both have the same number of unpaired
nucleotides [a(2)/3(O)] at the basis of the extra arm, but
Ser
tRNA Ser Y 4 i s m uch better serylated than tRNA Y2 (Fig. 2B).
However, this could also result from the difference of the size
and the shape of the variable loops. tRNASerY2 has a loop of
4 nucleotides (UAGC) while tRNASerY4 has only 3 nucleotides
(UCU). This view is supported by the fact that mutation of G47:A
to A47:A in tRNASerY4 (Fig. 1) made it behave like tRNASerY2
upon serylation (Fig. 2B, Y5), whereas the same mutation in
tRNA 5 " did not significantly affect serylation (Breitschopf and
Gross, unpublished).
Serylation of tRNA(Ser)Sec requires an appropriate T-/D-stem
loop interaction or three-dimensional structure. This is revealed
by the reduction of serylation resulting from mutations in the Dand T-stems of tRNA'5")5"* (Fig. 2A, X9, X10, XI1 and
unpublished data). Similar requirements were also observed for
tRNA 5 " (9).
Some of the cytoplasmic leucine tRNAs in eukaryotes have
also a long extra arm in an orientation [a(l)/3(2)] similar to that
of tRNA5", although their sequences are different from that of
tRNASer or tRNA(Ser)5ec. It is plausible that an interaction could
occur between the extra arm of leucine tRNA and the seryl-tRNA
synthetase, since there is no requirement of sequence specificity
in the extra arm for seryl-tRNA synthetase. The sequences in
the acceptor stems of these two tRNAs are different from each
other (4). However, the acceptor stem does not contribute
significantly to the differentiation between tRNA 5 " and other
tRNAs (9). This is also true in the case of t R N A ' 5 " ^ (Figs 1
and 2A, X8). The discriminator base A73 is highly conserved
in cytoplasmic leucine tRNAs (4), and is one of the identity
element of E. coli tRNA1*11 (37). As mentioned above, G73 is
an essential identity element for serine tRNAs. Hence,
mischarging of tRNA1*11 by seryl-tRNA synthetase will be
mainly avoided through the discriminator base. However,
tRNA(5er)See with A73 can still be weakly serylated under
particular conditions (not shown). This suggests that other
mechanisms may also exist to ensure the accuracy of
aminoacylation. A competiton of leucyl-tRNA synthetase for
tRNA1*11 in vivo might contribute to reduce the potential of
mischarging of tRNA1*11 by seryl-tRNA synthetase. In E. coli,
such a competition mechanism has been studied in some detail
(38,39). The difference in the tertiary structure has also been
proposed to be involved in the discrimination between tRNA 5 "
and tRNA1*11 both in prokaryotic and eukaryotic systems (5).
Our results presented here suggest that a long extra arm in
an appropriate orientation and the discriminator G73 are the major
identity elements of human tRNA(5er)Sec for serylation. The T/D-stem interaction plays an unspecific role by establishing the
overall tertiary structure, whereas the anticodon is not involved
in serylation of tRNA(Ser)Sec (not shown). This set of identity
elements is similar to that of tRNA 5 " (9). The known
cytoplasmic tRNAs s " from eukaryotes are very similar in
sequence, especially they all have a G73 discriminator base and
a similar extra arm of 14 nucleotides (4). The notion that G73
and a long extra arm in an approriate orientation are the major
identity elements and that there is no requirement for a specific
sequence in the extra arm suggests that all eukaryotic seryl-tRNA
synthetases use the same recognition mechanism.
ACKNOWLEDGEMENTS
We thank H.-D. Sickinger and Dr. T. Achsel for the synthesis
of deoxyoligonucleotides and Prof. H. Beier for critically reading
the manuscript. This work was supported by Deutsche
Forschungsgemeinschaft (SFB 165) and Fonds der Chemischen
Industrie.
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