© 7992 Oxford University Press
Nucleic Acids Research, 1992, Vol. 20, No. 22
5911-5918
Pseudouridine in the anticodon G^A of plant cytoplasmic
tRNATyr is required for UAG and UAA suppression in the
TMV-specific context
Karin Zerfass and Hildburg Beier*
Institut fur Biochemie, Bayerische Julius-Maximilians-Universitat, Biozentrum, Am Hubland,
D-8700 Wurzburg, Germany
Received September 7, 1992; Revised and Accepted October 20, 1992
ABSTRACT
We have previously isolated and sequenced Nicotiana
cytoplasmic tRNAT*r with G*A anticodon which
promotes readthrough over the leaky UAG termination
codon at the end of the 126 K cistron of tobacco mosaic
virus RNA and we have demonstrated that tRNATvr
with Q^A anticodon is no UAG suppressor. Here we
show that the nucleotide in the middle of the anticodon
(i.e., t 3 5 ) also contributes to the suppressor efficiency
displayed by cytoplasmic tRNATvr. A tRNATvr with GUA
anticodon was synthesized in vitro using T7 RNA
polymerase transcription. This tRNAT?r was unable to
suppress the UAG codon, indicating that nucleotide
modifications in the anticodon of tRNATvr have either
stimulating (i.e., * 3 5 ) or inhibitory (i.e., Q34) effects on
suppressor activity. Furthermore, we have shown that
the UAA but not the UGA stop codon is also efficiently
recognized by tobacco tRNAj^A> if placed in the TMV
context. Hence this is the first naturally occuring tRNA
for which UAA suppressor activity has been
demonstrated. In order to study the influence of
neighbouring nucleotides on the readthrough capacity
of tRNATyr, we have established a system, in which
part of the sequence around the leaky UAG codon of
TMV RNA was inserted into a zein pseudogene which
naturally harbours an UAG codon in the middle of the
gene. The construct was cloned into the vector pSP65
and in vitro transcripts, generated by SP6 RNA
polymerase, were translated in a wheat germ extract
depleted of endogenous mRNAs and tRNAs. A number
of mutations in the codons flanking the UAG were
introduced by site-directed mutagenesis. It was found
that changes at specific positions of the two
downstream codons completely abolished the
readthrough over the UAG by Nicotiana tRNATvr,
indicating that this tRNA needs a very specific codon
context for its suppressor activity.
INTRODUCTION
Natural suppression is the readthrough over mRNA termination
codons stimulated by tRNAs from wild-type cells. One of the
well known cases of natural suppression occurs during the
* To whom correspondence should be addressed
translation of tobacco mosaic virus (TMV) RNA which contains
two open reading frames separated by an in-frame UAG stop
codon (1). Readthrough over this internal stop codon generates
a 183 K polypeptide in cell-free extracts (2, 3) and in TMVinfected cells or protoplasts (4-6). The 183 K polypeptide is
supposed to be involved in viral replication because it has
considerable amino acid sequence homology to other viral RNAdependent polymerases (7, 8). It appears to be essential for
viability of the virus (9). We have previously isolated and
sequenced the two major cytoplasmic tyrosine tRNAs with G*A
anticodon from tobacco and wheat leaves which promote UAG
suppression in TMV RNA during in vitro translation in
reticulocyte lysate or wheat germ extract (10, 11).
Another example of natural suppression in plants has been
observed upon the expression of tobraviruses. These viruses
possess a bipartite genome packaged separately in rod-shaped
particles (12). RNA-1 encodes two open reading frames separated
by an UGA stop codon (13). The readthrough protein of about
190 K was detected in vitro (14, 15) and in vivo (16). The tRNAs
responsible for the suppression of the UGA codon in tobacco
rattle virus (TRV) RNA-1 have recently been isolated from
tobacco leaves. They were identified as chloroplast and
cytoplasmic tryptophan tRNAs. These two tRNAs have the
anticodon CmCA but suppress the UGA stop codon with
different efficiencies (17).
In both types of natural suppression mentioned above, a nonconventional base-pairing occurs at the first position of the tRNA
anticodon: a G:G mismatch in the case of tRNAjj!^ recognizing
the UAG and a Cm:A mismatch in the case of tRNAc^ A
suppressing the UGA stop codon. The mechanism of this
unorthodox codon reading is not clear. We have, however, shown
that tRNATyf with the hypermodified nucleoside queuosine (Q)
at the first position of the anticodon does not promote UAG
readthrough (11), emphasizing the importance of a G:G
interaction.
Pseudouridine (*) at the second position of the anticodon, a
unique feature of all eukaryotic cytoplasmic tRNAsTy, may
interact more firmly with adenosine than uridine, thus
strengthening the unconventional anticodon:stop codon
interaction. We have therefore studied the influence of * in the
anticodon of tRNAT>'r on its suppressor activity by in vitro
synthesis of tRNATyr with a GUA anticodon.
5912 Nucleic Acids Research, 1992, Vol. 20, No. 22
A recent report by Skuzeski et al. (18) has claimed that the
signal for readthrough over all three stop codons includes the
two downstream codons. These authors used an in vivo assay
in which suppression of a stop codon was coupled with the
transient expression of /3-glucuronidase (GUS) reporter genes in
tobacco protoplasts. In this system, the nature of the suppressor
tRNA responsible for the observed readthrough remains
unknown.
In order to find out whether it is tRNA 1 ^ which is able to
read all three stop codons and which requires a specific context
for suppression we have cloned the stop codon and parts of the
known flanking sequences derived from TMV RNA into the
middle of a zein gene from maize and investigated the expression
of the full-length zein protein in a tRNA-depleted wheat germ
extract supplemented with purified tobacco tRNATy. By
introducing mutations into the readthrough region, we found that
it is indeed tRNA 7 ^ which requires a specific codon context for
UAG suppression and that UAA but not UGA is also recognized
by tRNATy in the TMV-specific context.
MATERIALS AND METHODS
Enzymes and reagents
Restriction endonucleases, SP6 RNA polymerase and T4
polynucleotide kinase were from Boehringer, Mannheim. T7
RNA polymerase was purified as described (19). T7 DNA
polymerase sequencing kit was from Pharmacia LKB.
[14C]Methylated proteins as molecular weight markers for gel
electrophoresis and [35]methionine with a specific activity of 37
TBq/mmol were obtained from Amersham. Untreated wheat
germs were a gift from Synpharma GmbH, Augsburg.
Bacterial strains and plasmids
Escherichia coli JM109 and TGI were used as hosts for the
propagation of plasmid DNAs and M13mpl9 phages,
respectively. The recombinant plasmid used for site-directed
mutagenesis was pSP65-MLl, which carries a zein gene from
maize seedlings DNA (20) cloned into the BamHl and Pstl sites
of pSP65 vector DNA (17). The plasmid pDW27 encoding the
M1 RNA sequence flanked by a T7 RNA polymerase promoter
(21) was a gift from Dr. G.Krupp, Kiel.
Oligonucleotide-directed mutagenesis
Site-directed mutagenesis was performed according to Kunkel
(22). The 1218 bp long BamHVPstl fragment containing the zein
gene was first excised from pSP65-MLl DNA and cloned into
M13mpl9 RF-DNA. Synthetic oligonucleotides used to replace
zein-specific sequences around the internal TAG codon by the
TMV readthrough region (= pSP65-TMV) and for all further
mutated clones derived from this construct were synthesized by
the Gene Assembler Plus from Pharmacia LKB. The mutant
DNA was recloned into the pSP65 vector. The introduction of
the desired mutations was confirmed by direct sequencing of
plasmid DNAs (23).
Transcripton in vitro
In vitro transcription of the plasmid pSP65-TMV and its
derivatives by SP6 RNA polymerase was carried out as described
(17). For in vitro transcription with T7 RNA polymerase,
template DNA was prepared by BstNl digestion of the plasmid
pNtYlA-T7. The reaction mixture was phenol-extracted and the
DNA ethanol-precipitated. Preparative 'run-off transcription was
performed essentially as described by Milligan and Uhlenbeck
(24). Reaction mixtures contained in a total volume of 200 /tl:
10 ng template DNA, 40 mM Tris-HCl (pH 8.1), 6 mM
MgCl2, 1 mM spermidine, 5 mM dithiothreitol, 750 fiM each
NTP and 6 /ig T7 RNA polymerase. After incubation for 90 min
at 37°C, the reaction mixture was extracted once with
phenol/CHCl3/isoamylalcohol (25:24:1), once with CHC13/
iso-amylalcohol (24:1) and ethanol-precipitated in the presence
of 0.7 M NH4OAc. The transcripts were purified on a 12.5%
polyacrylamide/8M urea gel.
In vitro processing of tRNA precursors
Ml RNA was synthesized by T7 RNA polymerase using SnaBldigested pDW27 DNA as template (21). In vitro processing of
the pre-tRNA with purified Ml RNA was carried out in 0.5 M
NH4OAc, 50 mM Mg(OAc)2, 50 mM Tris-acetate, pH 8.0 at
45 °C overnight. Under these conditions 100% of the pre-tRNA
is converted to mature tRNA. The processed tRNA was
subsequently purified by gel electrophoresis. The accuracy of
cleavage by Ml RNA was confirmed by RNase Tl fingerprint
analysis of the transcript labelled with [a32P]GTP.
Fractionation and isolation of tRNAs
Preparation of tRNA from leaves of Nicotiana rustica followed
by fractionation of total tRNA on a BD-cellulose column was
performed as previously described and the tRNATyr-specific
fractions with UAG suppressor activity were further purified by
successive polyacrylamide gel electrophoreses (10). Total tRNA
from cells of Tetrahymena thermophila was isolated according
to Kuchino et al. (25). BD-cellulose fractions enriched in
tRNAGln with CUA anticodon (26) were used for in vitro
translation assays.
Preparation of aminoacyl-tRNA-synthetases
Wheat germ aminoacyl-tRNA-synthetase was isolated from wheat
embryos as described (17). Aminoacyl-tRNA-synthetase from
Tetrahymena was prepared according to Kuchino et al. (25).
Preparation of a mRNA- and tRNA-dependent wheat germ
extract
A wheat germ cell-free extract depleted of endogenous mRN As
and tRNAs was prepared essentially as described by Pfitzinger
et al. (27). The protein concentration was 17 mg/ml.
Translation in vitro
Translation in the tRNA-depleted wheat germ extract was carried
out in a total volume of 10 /tl containing 30% (v/v) wheat germ
extract, 10% (v/v) wheat germ initiation factor solution, 20 mM
HEPES-KOH (pH 7.5), 2 mM dithiothreitol, 1 mM ATP, 20 fM
GTP, 8 mM creatine phosphate, 40 /ig/ml creatine
phosphokinase, 50 /iM spermine, 2.5 mM Mg(OAc)2, 150 mM
KOAc, 70 /iM each of 19 L-amino acids (without methionine),
15 kBq/ml [35S]methionine and 30-50 /tg/ml template RNA.
tRNA concentrations are given in the legends.
Analysis of translation products
Proteins were analysed by electrophoresis in 15% polyacrylamide
slab gels containing SDS (28). Gels were fixed overnight,
fluorographed as described (29) and exposed to RX Fuji X-ray
film at -80°C. To quantify the relative amounts of radioactivity
in the different bands, the fluorograms were evaluated
densitometrically using an Elscript 400 scanner.
Nucleic Acids Research, 1992, Vol. 20, No. 22 5913
Wrl
pSP6J-MLl:
pSP«5 - TMV : -AAC-CAcfga-ACA-CAA-TAC-CAA-riA-CAlj-TTT-CTGpSP«5 - TMV,: -AAC^AcfGGA-ACA-CAA-TAC-CAA-TTA-CAGJ-TTT-CTG-
AUO
UAG
UAG UGA
nin-off trtnscnpt:
1246 nt
UKpolypepdde:
132 u
29 Krcidthroughprotein:
32 K leadthrough protein:
265 « l
292 M
Figure 1. Structures of expression vector pSP65-MLl and its derivatives. A 1.2 kb fragment from Zea mays DNA, harbouring a zein gene, was cloned into the
Bamlil and Psll sites of the SP6 RNA polymerase-specific vector pSP65. The coding region of the zein gene is indicated by a hatched box, the flanking sequences
originating from Z mays genome are marked by thick lines. The arrow points to the transcription initiation site. Part of the sequences surrounding the TAG stop
codon in the middle and at the end of the zein gene are shown. Two constructs (i.e., pSP65-TMV and pSP65-TMV,) contain a TAG and a TAC codon, respectively,
and a total of six codons flanking the leaky UAG codon in TMV RNA. In vitro translation of the run-off transcript results in the synthesis of a 14 K protein and—in
the presence of an appropriate UAG suppressor tRNA—two readthrough proteins of 29 and 32 K.
RESULTS
Suppression of the TMV-specific UAG stop codon in the
construct pSP65-TMV
The identification of tobacco tRNATyr as UAG suppressor (10)
raised the question whether this tRNA is able to recognize not
only the leaky UAG stop codon in TMV RNA but also potential
stop codons occuring in-frame in the coding region of cellular
mRNAs. To examine this possibility we selected the zein gene
ML1 from Zea mays with an internal UAG codon (20). In-frame
stop codons have been found in a number of storage protein genes
(30-33) but it is open until now whether these stop codons are
functional as terminator elements.
The zein gene ML1 was cloned into the SP6 RNA polymerasespecific vector pSP65. The resulting clone was named
pSP65-MLl. In vitro transcription with SP6 RNA polymerase
yields a run-off transcript of 1246 nt (Fig. 1). A protein of 14000
(14 K) is expected if translation terminates at the internal UAG
stop codon. Readthrough over this first stop codon results in the
synthesis of a 29000 (29 K) and a 32000 (32 K) protein, if the
stop codon at the end of the zein cistron is also suppressed
(Fig. 1).
Translation of the in vitro synthesized transcripts was carried
out in a wheat germ extract depleted of endogenous mRNAs and
tRNAs according to Pfitzinger et al. (27). Fig. 2 (lanes a, d and
g) shows that indeed no product was made in this extract in the
absence of added mRNA and tRNA. When the pSP65-MLl
ML1
TMV
TMV,
97
69
46
32K-
30
14.3
14K —
Figure 2. Effect of Tetrahymena UAG-reading tRNAcu^ and Nicotiana
tRNAj!£A on translation of three different transcripts. In vitro transcripts derived
from pSP65-MLl, pSP65-TMV and pSP65-TMV, (illustrated in Fig. I) were
synthesized by SP6 RNA polymerase and translated in a wheat germ extract
depleted of endogenous mRNAs and tRNAs. Nicotiana \RNALQ$'A (lanes b, e
and h) and Tetrahymena tRNA^jjA (lanes c, f and i) were added to the translation
mixtures at concentrations of 50 /ig/ml. Lanes a, d and g show the control
incubations in the absence of added mRNA and tRNA. The [35S]methioninelabelled proteins were separated on a 15% denaturing gel. [l4C]Methylated
proteins ranging in size from 14.3 to 97 K were added as mol. wt. markers at
the right side of the gel. The major translation product of 14 K and the readthrough
proteins of 29 and 32 K are indicated at the left.
5914 Nucleic Acids Research, 1992, Vol. 20, No. 22
TMV
TMV«
TMVs
TMVB
TMVg
TMVg
TMVio
TMV11
TMVn
TMV13
TMV14
TMV 15
TMV I 6
97
69
46
30
29K
-
14 3
14K- '
Figure 3. Readthrough over the UAG stop codon in transcripts derived from pSP65-TMV by Nicotiana t R N A j ^ . In vitro synthesized transcripts pSP65-TMV4
to pSP65-TMV,6 carrying mutations in the TMV-specific readthrough region 5' or 3' of the UAG stop codon (indicated in Table 1) were translated in wheat germ
extract in the absence (left lane) or in the presence of 50 ^g/ml Nicotiana tRNAjj!^ (right lane). [l4C]Methylated proteins were added as mol. wt. markers at the
right side of the gel. The major translation product of 14 K and the readthrough protein of 29 K are indicated at the left.
transcript was translated in the presence of Nicotiana tRNAT>"\
a major product of 14 K was synthesized but no readthrough
protein was visible (Fig. 2, lane b). When Tetrahymena
tRNAGln with CUA anticodon had been added instead of
tRNAJyr, the 32 K and to a lesser extent the 29 K readthrough
proteins were synthesized (Fig. 2, lane c).
A comparison of sequences from a number of plant viral RNAs
which harbour leaky UAG stop codons reveals that they have
similar sequences around the termination codon, preferentially
at the 3' side (1, 34—39) suggesting that these nucleotides might
be important for suppression. We therefore inserted a sequence
comprising three codons on each side of the leaky UAG stop
codon of TMV RNA (1) into the zein gene such that the
corresponding nucleotides flanking the internal UAG codon in
the zein gene were replaced. This construct was called
pSP65-TMV (Fig. 1). The protein pattern observed in the
presence of the pSP65-TMV transcript and Tetrahymena
tRNAcuA (Fig. 2, lane 0 was essentially the same as seen with
the pSP65-MLl transcript (lane c). However, translation of the
pSP65-TMV transcript in the presence of Nicotiana tRNA 7 ^
now yielded the 29 K readthrough protein (Fig. 2, lane e) which
was not visible with the pSP65-MLl transcript (lane b). A protein
of the same size was synthesized when the transcript used for
translation (i.e., pSP65-TMV]) contained a tyrosine codon
instead of the UAG stop codon (Fig. 2, lane h), indicating that
the 29 K protein is indeed the expected readthrough protein. From
these results we conclude that plant tRNATyr needs a specific
context for efficient UAG suppression and that this context does
not appear to include more than three codons on either side of
the UAG. Apparently this does not apply to Tetrahymena
tRNAcu^ which reads the UAG stop codon in three different
surroundings, i.e., the internal UAG in the zein gene, the UAG
in the TMV context and the UAG at the end of the zein cistron
(Figs. 1 and 2). However, it cannot be ruled out that UAG
suppression by this tRNA is modulated by yet unidentified
surrounding nucleotides.
Influence of the codon context on the UAG suppression by
Nicotiana tRNAT>r
In order to localize the nucleotides important for UAG
readthrough by tobacco tRNATyr, we first exchanged the codons
5' and 3' of the UAG in pSP65-TMV to GAC in pSP65-TMV4
and GGA in pSP65-TMV5, respectively (Table 1). These
Table 1. Readthrough over the UAG stop codon by Nicotiana tRNAj,^ as a
function of surrounding nucleotides.
Transcript
Readthrough region
Readthrough
pSP65-MLl
pSP65-TMV
pSP65-TMV4
pSP65-TMV5
pSP65-TMV6
pSP65-TMVg
pSP65-TMV9
pSP65-TMV,0
pSP65-TMVn
pSP65-TMVl2
pSP65-TMV13
pSP65-TMV,4
pSP65-TMV,5
pSP65-TMVl5
-CAA-CAA-CAA-UAG-CUG-CAA-CAG-GGA-ACA-CAA-UAG-CAA-UUA-CAG-GGA-ACA-GAC-UAG-CAA-UUA-CAG-GGA-ACA-CAA-UAG-GGA-UUA-CAG-GGA-ACA-CAA-UAG-CAA-GGU-CAG-GGA-ACA-CAA-UAG-GAA-UUA-CAG-GGA-ACA-CAA-UAG-CGA-UUA-CAG-GGA-ACA-CAA-UAG-CAG-UUA-CAG-GGA-ACA-CAA-UAG-CAC-UUA-CAG-GGA-ACA-CAA-UAG-CAA-CUA-CAG-GGA-ACA-CAA-UAG-CAA-GUA-CAG-GGA-ACA-CAA-UAG-CAA-UCA-CAG-GGA-ACA-CAA-UAG-CAA-UUG-CAG-GGA-ACA-CAA-UAG-CAA-UUA-GAG-
1.5
100
82
0
10
1.5
5
70
4
69
4.5
30
6
39
Compilation of data shown in Figs. 2 and 3. Readthrough activity was defined
as 100% for the translation of pSP65-TMV transcript, containing the unmutated
read-through region from TMV RNA. Substituted nucleotides are underlined.
codons were chosen in the first place because they flank the leaky
UAG in Moloney murine leukaemia virus (Mo-MuLV) RNA
(40). For this virus it has been shown that a glutamine residue
is inserted at the termination codon in the gag-pol precursor
protein (41) and that the tRNA responsible for this UAG
suppression is very likely tRNAGln with UmUG anticodon (42),
a tRNA quite different from tRNAT5"\
The vector-DNA containing the authentic readthrough region
of TMV-RNA (i.e., pSP65-TMV) and the two clones carrying
mutations in this region (i.e., pSP65-TMV4 and TMV5) were
transcribed by SP6 RNA polymerase and translated in wheat germ
extract. Fig. 3 clearly shows that exchange of the codon 5' to
the UAG had little influence on the readthrough capacitiy of
Nicotiana tRNATyf whereas exchange of the codon immediately
3' to the UAG completely abolished suppression. The next
downstream codon, if replaced by GGU in pSP65-TMV6 (Table
1), also had a strong negative effect on suppressor activity
(Fig. 3).
In translation experiments using the pSP65-TMV transcript as
template we measured about 3 0 - 3 5 % readthrough protein
synthesis (Fig. 3). This value was defined as 100% in all further
Nucleic Acids Research, 1992, Vol. 20, No. 22 5915
TMV
TMV 2
TMV 3
Sspl
BslNI
if--'' Jt*
+ F>Vf77's
WV*////////////?
EcoRI
**•
i GGCAGA
nnr* A n A1 '
Figure 4. Ability of Nicotiana tRNA^' A to read UAG and UAA but not the
UGA termination codon. The in vitro synthesized transcripts pSP65-TMV,
pSP65-TMV2 and pSP65-TMV3 carrying the UAG, UAA and UGA stop codon,
respectively, in the TMV context were translated in wheat germ extract in the
absence (lanes a) or in the presence of 15 jig/ml Nicotiana tRNAjj!^ (lanes b).
in vitro translation assays with mutated transcripts (Table 1). It
is slightly higher than the readthrough activity of 25 % measured
when full-length TMV RNA was translated in reticulocyte lysate
in the presence of purified tobacco tRNATyT (10).
In the following studies we concentrated on pinpointing the
effects of single nucleotide exchanges in the two downstream
codons (Table 1, Fig. 3) which can be summarized as follows:
G in the first position of the 3' codon is absolutely inhibitory
for UAG suppression by Nicotiana tRNATyr, G in the second
position of the 3' codon reduces readthrough activity to 5%, C
in the third position has a similar inhibitory effect but G in the
third position of the 3' codon still allows 70% suppressor activity
as compared with the wild-type transcript. Substitution of the two
uridines in the first positions of the second 3' codon by a C leads
to a reduction of 30 and 70%, respectively, whereas a G in the
third position of this codon has a dramatic effect on suppression
(Table 1, Fig. 3). These observations are consistent with the
results obtained by Skuzeski et al. (18) who found that the
sequence CARYYA at the 3' side of the stop codon is the most
favourable context for stop codon suppression in tobacco
protoplasts.
UAG and UAA but not the UGA stop codon are recognized
by Nicotiana tRNA1*1"
For the recognition of UAG by tRNATyr with G*A anticodon
an unconventional G:G base-pairing in the first position of the
anticodon has to be postulated. We had formerly ruled out that
tRNATyr is able to read the UAA codon (involving a nonclassical A:G interaction) since we had not observed suppression
of the UAA codon at the end of the 183 K cistron of TMV or
any readthrough over the UAA codon at the end of a-globin
mRNA (11). However, in the light of our new findings that
tRNATyT requires a specific codon context for efficient UAG
suppression, we re-evaluated these studies using a template in
which the UAA codon was inserted into the TMV-specific context
in the pSP65 vector (pSP65-TMV2). In parallel we constructed
a template in which the UAG was replaced by UGA
(pSP65-TMV3). Both transcripts were translated in wheat germ
extract in the presence of tobacco tRNATyr. Using the transcript
containing the UAA stop codon, readthrough synthesis was
Figure 5. Structure of expression vector pNtYlA-T7. The plasmid pNtYl which
harbours an intron-containing tRNATyr gene on a 3 kb EcoRI fragment from
Nicotiana rustica DNA in the plasmid pUC19 (71) served as original plasmid
DNA from which a 255 bp SspVAccl-fragment was excised and ligated into
M13mpl9 RF-DNA cleaved with Sspl and Smal. Subsequent mutagenesis with
three different ohgonucleotides resulted in an intron-less tRNATyr gene flanked
by the T7 RNA polymerase promoter and a sequence enhancing initiation of
transcription by the latter at the 5' side and a BsiNl restriction site downstream
of the tRNA gene. The mutated DNA fragment was recloned into the SspVEcoRisites of pUC19 DNA. This clone was named pNtYlA-T7. The coding region
of the tRNATyr gene is indicated as a hatched box, the flanking sequences
originating from the Nicotiana genome are marked by thick lines. Run-off
transcription of the BwNI-digested pNtYl A-T7 DNA yields a pre-tRNATyr with
a mature CCA 3' end and the flanking sequence pppGGCAGA at the 5' side.
observed, albeit with a reduced rate of about 50% as compared
with the template carrying the UAG codon. The UGA stop codon
in the TMV context, however, was not suppressed at all by
tRNATyr (Fig. 4). These experiments were performed with
highly purified, sequenced tRNATyT obtained after twodimensional gel electrophoresis, thus excluding any traces of a
contaminating tRNA. Since the tRNA material of this fraction
was limited we used only 15 jig/ml (instead of 50 /tg/ml) final
concentrations in the translation experiments which explains the
overall reduced suppressor activity.
Suppressor activity of tRNAT>T depends on the
pseudouridine modification (*35) in the anticodon
Since an unconventional base-pairing has to be postulated for the
interaction of tRNAl^A with either of the two stop codons
UAG and UAA, we reasoned that pseudouridine in the second
position of the anticodon may interact with adenosine more firmly
than generally expected. To examine this possibility we
constructed a clone containing a Nicotiana intron-less tRNATyT
gene. Run-off transcription of the ftttNI-digested plasmid DNA
pNtYlA-T7 yields a transcript of 82 nucleotides length which
has a mature CCA 3' end and a six nucleotides long 5' flanking
sequence (Fig. 5). In order to generate a transcript with a mature
5' end we used two different strategies: We removed the 5'
flanking sequence in vitro with Ml RNA, or we incubated the
pre-tRNA in a wheat germ S23 extract, depleted of endogenous
tRNAs by DEAE-cellulose chromatography. This extract had
retained most of its processing and modifying activities. The
transcript derived from pNtYlA-T7 (Fig. 5) was incubated for
60 min at 30°C in the tRNA-depleted extract and mature
tRNATyT with GUA anticodon was isolated from a preparative
gel. A pseudouridine in the middle of the anticodon (^35) cannot
be formed since * 3 5 synthase uses only intron-containing pretRNAs as substrate (43-45). However, a number of other
modified nucleotides typical for tRNATyT (10) like m2G at
position 10, m^G at position 26 and partially m'G at position
37 were introduced.
Bare and Uhlenbeck (46) have shown that substitution of ty35
by an U in yeast tRNAj|^ increased the Km for aminoacylation
5916 Nucleic Acids Research, 1992, Vol. 20, No. 22
29K»-
14
G:G.
K»~
Figure 6. Suppressor activity of tRNATyr as a function of nucleotide modification
in the anticodon. The transcript pSP65-TMV containing the authentic readthrough
region of TMV RNA was translated in wheat germ extract in the presence of
15 /ig/ml and 50 /ig/ml, respectively, Nicotiana t R N A j ^ (b, e) and 20 /ig/ml
in vitro synthesized t R N A j ^ , processed and partially modified in wheat germ
extract (c) or processed by Ml RNA (d). Lane a shows the protein pattern in
the absence of added tRNA.
only by a factor of two. In accordance with these results we found
that fully modified t R N A j ^ isolated from tobacco leaves and
partially modified tRNA 7 ^ with GUA anticodon were both
efficiently charged with tyrosine by an unfractionated wheat germ
synthetase preparation. Furthermore we confirmed that precharged synthetic tRNATyr generated by Ml RNA transferred
tyrosine into proteins synthesized in wheat germ extract, albeit
at a reduced rate as compared to fully modified Nicotiana
tRNA 7 ^ (not shown). A similar observation about the properties
of tRNA lacking modified nucleosides has been made by
Samuelsson et al. (47).
We then translated the pSP65-TMV transcript containing the
authentic readthrough region of TMV in the presence of fully
modified tRNATyr with G^A anticodon isolated from tobacco
leaves (Fig. 6, lanes b and e), and in parallel we added
unmodified tRNA 1 ^ with GUA anticodon generated by in vitro
transcription and Ml RNA processing (Fig. 6, lane d) or partially
modified tRNATyr with GUA anticodon processed in the wheat
germ extract (Fig. 6, lane c) to the translation mixture. Only
tRNATyr with G^A anticodon stimulates the synthesis of the
29 K readthrough protein as seen in Fig. 6. The same results
were obtained when the pSP65-TMV2 transcript, containing the
UAA stop codon in the TMV context, was used as template (not
shown).
DISCUSSION
Cytoplasmic tRNAc^, a natural suppressor tRNA operating in
higher plants, has been shown to exhibit a number of interesting
features: i) base modifications in the first and in the second
position of the anticodon have either inhibitory or stimulating
effects on UAG suppression, ii) the two termination codons UAG
and UAA but not UGA are suppressed and iii) suppressor activity
strictly depends on a specific codon context.
G x Q.,n
Figure 7. Schematic presentation of hypothetical interactions between noncomplementary purine bases. In all models the nucleoside of the anticodon is
shown in the syn configuration (isomerization about the glycosyl bond of the
nucleoside). G G base pairs involving two H bonds can be formed with a G in
the rare tautomeric enol-imino form (b) or with a G in the major tautomeric ketoamino form (a) requiring a small deviation in the glycosyl bond angle. A:G base
pairs can be formed with the A in either the protonated (c) or the imino form
(d). Any type of interaction with queuosine in the syn conformation is impaired
by the space-filling cyclopentene-diol side chain (e).
All eukaryotic cytoplasmic tRNAsTyr contain a pseudouridine
(*35) in the middle of their anticodons (48). In order to study
the effect of this modified nucleoside on UAG readthrough, we
consequently had to synthesize tRNATyr with a GUA anticodon
in vitro. The pre-tRNA transcript with a 5'-flanking sequence
was processed with Ml RNA, the catalytic subunit of RNase P
(49), or was incubated in a wheat germ extract to yield a tRNA
of mature length. The generation of tRNATyr in this extract has
the advantage that a number of modifications are introduced into
the in vitro synthesized pre-tRNA which are present in Nicotiana
tRNATyr (50). The conversion of U35 to * 3 5 is excluded because
the pre-tRNA used here contains no intron which is a prerequisite
for ^35 synthesis (43—45). The completely unmodified and the
partially modified tRNATyr, respectively, with GUA anticodon
are both no UAG (Fig. 6) or UAA suppressors, indicating that
^35 plays an important role in the interaction of tRNA J$'A with
either UAG or UAA. Ward and Reich (51) have shown that
alternating A:^ polynucleotides have a dramatically higher
melting temperature of 58°C as compared with 32°C of
corresponding A:U-polymers. Recent observations by Johnson
and Abelson (43) with an undermodified UAA suppressor also
support the idea of a strong A:* interaction. These authors found
Nucleic Acids Research, 1992, Vol. 20, No. 22 5917
a significant decrease of UAA suppressor activity displayed by
yeast tRNAj^ A when the * in the anticodon was changed to U.
Pseudouridine can form a classical base pair with A in the anti
and syn configuration making it more versatile in its hydrogen
bonding interactions. Furthermore, an extra NH moiety is created
when the ribose is shifted from N) in U to C5 in ^ . This new
proton presents a site for the potential formation of a third
hydrogen bond. It was shown by Griffey et al. (52) that the
tertiary interaction of ^ 3 2 in Escherichia coli tRNAPhe with
2'-hydroxyl groups in nearby ribose units stabilizes the
conformation of the anticodon loop. We can only speculate that
a similar stabilization is achieved by ^ 3 5 in the codon:anticodon
interaction.
Grosjean et al. (53) have studied the relative stabilities of
complexes between tRNAs complementary in their anticodons.
They determined about the same low lifetimes for G:U and G:^
pairs in the middle of the anticodon. Since tRNAj^A does not
recognize the UGA stop codon which would involve a G:^
interaction, but reads UAG and UAA (Fig. 4), involving an A:¥
pair, we consequently conclude that the latter indeed contributes
to the unconventional base pairing observed for tRNATyr.
The first position of the tRNAT>'r anticodon is often occupied
by a queuosine (Q), a highly modified G derivative (54). It has
been shown earlier that only tRNATyr with G*A anticodon but
not with Q^A anticodon can read the UAG codon (11, 55).
Interestingly, tobacco and wheat leaves contain exclusively
j^A whereas in wheat germ about 85% of tRNA 1 ^ has a
anticodon (11, 56). Hence, variations in the extent of Qmodification in the respective tRNAs appear to modulate UAG
suppression by tRNAsT>'r. This does not apply to the content of
undermodified tRNAT>'r with respect to the *-modification since
virtually all eukaryotic tRNAsT>T carry a * 3 5 (48) which
obviously also enhances the interaction of tRNATyr with the
normal tyrosine codons.
We have shown that tRNAj^ A recognizes not only the UAG
but also the UAA codon if placed in the TMV context (Fig. 4).
This implies that we have identified the first naturally occurring
UAA suppressor tRNA in plants and altogether in any organism
of prokaryotic or eukaryotic origin. There are, however, a
number of reports which indirectly point to the existence of UAA
suppressor tRNAs in plants, yeast and vertebrates (9, 57 — 59).
Most relevant for our studies is the work from Ishikawa et al.
(9). These authors have established an in vitro transcription
system to produce infectious TMV RNA from a cloned cDNA
copy. Transcripts from cDNAs mutagenized at or near the leaky
UAG stop codon were assayed for infectivity in tobacco plants.
It was found that i) transcripts coding for a defective 183 K protein
were not infectious, emphasizing that the readthrough protein is
essential for the virus and that ii) replacement of the UAG by
an UAA codon retained the infectivity, indicating that readthrough
over the internal UAA had in fact occurred. As shown here,
tRNATyr is the most likely candidate to read that UAG and the
UAA replacing it in vivo.
At present we cannot explain the unconventional G*A:UAG
and G^A:UAA interaction which is not in accordance with the
wobble hypothesis. Considering the potential strong bond of the
A:^ pair, the 'two out of three' reading, i.e., interaction between
only two base pairs (one A:U and one A:^ pair) could apply
here (60). However, since the G^A anticodon recognizes the
UAG codon whereas the Q^A anticodon does not, we conclude
that the G in the first position of the anticodon must play a
selective role in stop codon suppression. We present two
alternative models for the putative purine base pairs based on
investigations by Topal and Fresco (61, 62) and Brown et al.
(63). In all cases, the G of the anticodon is drawn as the syn
isomer which occurs with a frequency of 10"' (61, 62),
whereas the A and G, respectively, of the codon is presented
either in the rare tautomeric form (Fig. 7b, d), not favoured under
physiological conditions (frequency = 10~4 to 10~5), or in the
major keto/amino tautomeric form (Fig. 7a) and a minor
tautomeric form in which the A is protonated (Fig. 7c). The G:G
interaction shown in Fig. 7a results in a slight displacement of
the glycosidic bond. A similar distortion occurs in the wobble
base pairs proposed by Crick (64). The possibility of a syn
conformation in the first position of the anticodon has been
discussed by Jank et al. (65) to explain the recognition of all four
valine codons by tRNA^c- The physical impossibility of an
interaction between Q in the syn form and a G (Fig. 7e) explains
the inability of tRNAjj;A to read UAG codons as mentioned
above.
A further striking feature of plant tRNATyr is its requirement
of a specific mRNA codon context for efficient suppression. The
UAG in the original transcript pSP65-MLl, containing a zein
pseudogene, was very poorly suppressed by tRNATyr but the
UAG in the transcript pSP65-TMV, harbouring the authentic
readthrough region of TMV RNA, was bypassed with an
efficiency of about 30% (Figs. 2, 3). Substitution of codons or
single nucleotides 5' and 3' of the UAG revealed a major
influence of the two downstream codons (Table 1), in agreement
with recent studies by Skuzeski et al. (18). These authors had
investigated readthrough over the UAG as a function of transient
expression of /3-glucuronidase reporter genes in tobacco
protoplasts. They found that the two 3' codons CAR YYA
conferred leakiness to all three stop codons. Our results strongly
imply that it is cytoplasmic tRNATyr which reads the UAG and
UAA stop codon in the TMV context, but that UGA must be
recognized by another tRNA since this codon is not suppressed
by tRNATy (Fig. 4).
We have recently identified tobacco chloroplast and
cytoplasmic tRNAc^fA as being responsible for the readthrough
over the UGA codon in RNA-1 from tobacco rattle virus (TRV)
and have shown that tRNATrP does not suppress the UGA at the
end of the /3-globin mRNA (17). Preliminary experiments have
revealed that the UGA codon in the TMV context is in fact
efficiently read by tRNAaS?A> indicating that this tRNA is a
likely candidate for the observed readthrough in vivo.
Little data exist until now about the effects of codon context
on suppression of nonsense codons in higher eukaryotes.
Principally one has to consider the competition between the
release factor normally terminating protein synthesis and the
suppressor tRNA. As far as codon context is involved, it appears
that only weak suppressors are dependent on a favourable
nucleotide sequence surrounding the stop codon whereas strong
suppressors are not (66, 67).
Angenon et al. (68) have made an analysis of the stop codon
context in plant nuclear genes. The most noticeable feature is
the finding of an underrepresentation of A and G at the - 1 and
- 2 position, respectively, and a very low percentage of C at
the 3' side of the termination codon. The nucleotides 5' and 3'
of the leaky UAG in TMV RNA are A and C, respectively
(Fig. 1), suggesting that this context might be deleterious for the
release factor. However, since the context required by tRNA7^
includes at least six downstream nucleotides, other features have
to contribute additionally to nonsense codon suppression.
5918 Nucleic Acids Research, 1992, Vol. 20, No. 22
Considering the fact that tRNA7^' needs the minimum
sequence of CAAUUA at the 3' side of the UAG stop codon,
we postulate that at least one other UAG suppressor tRNA must
exist in plants since, for example, the leaky UAG stop codon
in potato leaf roll virus is followed by GUAGAC (69) and by
GGGGGC in carnation mottle virus RNA (70).
ACKNOWLEDGEMENTS
We are grateful to Professor H.J.Gross for stimulating discussions
and for critical reading of the manuscript. We thank Dr. G.Feix
(Freiburg) for providing the clone pMLl and Dr. G.Krupp (Kiel)
for a gift of the plasmid pDW27. This work was supported by
a grant from the Deutsche Forschungsgemeinschaft to H.B.
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