(Received for publication, December 27, 1994; and in revised form, March 29, 1995)
From the
The importance of 2`-OH groups of codons for binding of cognate
tRNAs to ribosomal P and A sites was analyzed applying the following
strategy. An mRNA of 41 nucleotides was synthesized with the structure
C The transfer of the genetic message into the amino acid sequence
of a distinct protein is based on the ribosomal decoding process, which
occurs at the ribosomal P site during initiation and at the A site
during elongation. Our knowledge concerning the mechanism of the
decoding processes is sparse. Two models have been suggested. The
prevailing model, which has been more or less generally accepted over
the last 15 years, assumes that the discrimination energy responsible
for the accuracy of the decoding process is derived from the
stabilities of base pairing during codon-anticodon interaction. Studies
on lifetimes of anticodon-anticodon interactions in solution revealed
that the selection accuracy according to this model could hardly be
better than 1:10 (one incorrect selection per 10 correct ones) and
certainly not better than 1:100(1) . Since ribosomes select the
amino acids with a precision around 1:1000(2) , it is clear
that this experimental model does not properly reflect the ribosomal
decoding process. These findings led to the suggestion that the
discrimination energy inherent to base pairing between codon and
anticodon has to be exploited more than once(3) , and a
possible mechanism for this was provided by proofreading
theories(4, 5) . However, aqueous conditions may
not apply for the decoding center, which may provide an environment
where the energetics of the interaction are stronger and more
discriminating. A detailed model was suggested about 10 years
ago(6) , according to which the ribosomal decoding center
recognizes the codon-anticodon duplex in the same way as does the
active center of an enzyme, i.e. it recognizes the correctness
of the stereochemistry of the partial Watson-Crick structure formed by
the codon-anticodon duplex. This model implies that the sugar-phosphate
backbone of the codon-anticodon is recognized and is only bound if
correctly positioned, thus contributing to the discrimination energy,
which could be much larger than that predicted by the previous model. A decisive experiment to discriminate between the two models would
be a comparison of ribo-codons and 2`-deoxyribo-codons during tRNA
binding, since base pairing in RNA-RNA duplexes is almost identical to
that in DNA-RNA hybrids, which also adopt an overall A form typical for
RNA duplexes. However, the sugar puckering in the DNA strand of hybrids
is quite different from that of RNA duplexes(7) . The first
model predicts that DNA codons would be accepted just as well as RNA
codons, whereas according to the second model the ribosomal decoding
center should sharply discriminate between DNA codons and RNA codons. It has been shown that single-stranded DNA could not be translated
under normal
conditions(8, 9, 10, 11, 12) .
Poly(dT), in contrast to poly(U) is not accepted as mRNA in enzymatic
translation systems derived from the eubacteria Escherichia coli(11, 12) as well as from eukaryotic wheat germ
and rabbit liver; only yeast ribosomes show some spurious
activity(13) . In the absence of ribosomes the tRNA However,
experiments with homopolymeric mRNA such as poly(dT) could not
distinguish between effects of ribosome-mRNA interactions inside and
outside of the decoding region. Precise estimation of codon effects at
A, P, and E sites in the presence of homopolymeric mRNA is problematic
as well. Here we show with a heteropolymeric mRNA containing one
deoxyribo-codon that the tRNA binding to the A site depends on the
presence of the 2`-OH groups of the corresponding codon, in contrast to
tRNA binding to the P site. Furthermore, the A-site binding is impaired
when the upstream P site displays a deoxyribo-codon. Likewise, the
P-site binding is reduced when the upstream E site contains a
deoxyribo-codon. The results argue strongly in favor for the second
model, which suggests that the decoding center at the A site recognizes
the partial Watson-Crick structure formed by codon-anticodon
interaction. tRNA Tightly coupled 70 S ribosomes were isolated from midlog-phase
cells of E. coli, strain D10 (RNase 1
Table 1presents the mRNAs used in this study. They all
have the general structure C
In the first series of experiments we determine the binding of N-acetyl-Phe-tRNA
A strikingly different picture is found
when AcPhe-tRNA is bound to the A site (Table 2, experiment 3).
In the presence of EFV-mRNA (ribo-codon for Phe) the binding value is
the same as that found for P-site binding. In contrast, when a
deoxyribo-codon is displayed at the A site, the total binding is
reduced to one third, half of it being present at the A site.
Considering the A-site fractions found in the presence of UUC or dUdUdC
(2.55 and 0.5 pmol, respectively), the lack of the 2`-OH groups in the
A-site codon reduces the binding to this site by a factor of 5. Experiment 3 shows yet another detail. The low AcPhe-tRNA binding to
the A site in the presence of a deoxyribo-codon is much lower than the
binding to nonprogrammed P sites (1.0 versus 2.2 pmol). This
means that the tRNA In the next experimental series we bind an
AcVal-tRNA to P or A sites displaying a Phe codon in the ribo- or
deoxyribo-form upstream at E or P sites (Table 3). The binding
values to nonprogrammed (experiment 1) or programmed P sites
(experiment 2) are relatively low. The low binding can be explained by
the observations that nonprogrammed ribosomes have a low intrinsic
affinity for tRNA
Notably, the
binding to programmed P sites is reduced by more than 50% when the
upstream E-site codon is in the deoxy-form, i.e. P-site
binding to ribo-codons is impaired when the codon at the E site lacks
the 2`-OH groups but not when the deoxyribo-codon is at the A site (Table 2). An unexpected result is found in experiment 3 (Table 3). A 5-fold increase of binding to the A site over that
of the P site was observed in the presence of the EFV-mRNA (1.75 and
0.36 pmol, respectively). A similarly strong increase was already
reported earlier, when the codon was positioned in the middle of a
heteropolymeric mRNA (20) as is here the case. Surprisingly, if
a deoxyribo-codon is located at the P site, the A-site binding directed
by a ribo-codon drops by about 9-fold (1.75 and 0.2 pmol,
respectively). The decoding center at the A site thus recognizes a
deoxy-codon at the adjacent upstream P-site codon as does the P-site
center with the upstream E-site codon, but the A site reacts in a much
more sensitive manner, as indicated by the more strongly restricted
binding. The binding experiments with AcPhe-tRNA to A and P sites
depending on a UUC or dUdUdC codon were repeated with the MVF- and
MV(dF)-mRNA, respectively. The same results were found as those
demonstrated in Table 2obtained in the presence of EFV- or
E(dF)V-mRNA, respectively (data not shown). It follows that the
observed effects are solely due to the presence or absence of the 2`-OH
groups at the corresponding codon and are not modulated by different
context nucleotides. As an example conducted with the MVF/MV(dF) pair
of mRNAs, saturation curves of AcPhe-tRNA are shown in Fig. 1, A and C. In the absence of deacylated
tRNA
Figure 1:
Binding of AcPhe-tRNA to the A and P
sites of 70 S ribosomes programmed with either MVF or MV(dF) templates. A and C, saturation curves; C and D, the corresponding Scatchard plots(21) :
A tRNA binds to the ribosomal P site totally independently of
the presence of 2`-OH groups in the cognate codon at this site (Table 2, experiment 2). The binding is still significant even in
the absence of any codon (Table 2, experiment 1) but is severely
reduced in the presence of a near- or noncognate codon at the P site
indicating codon-anticodon interactions at the P
site(20, 22, 23) . The binding to the P site
is also not affected if the adjacent downstream codon at the A site is
of the 2`-deoxyribo-form (Table 2, experiment 3; see
``Results''), but is unexpectedly impaired if the adjacent
upstream codon at the E site lacks the 2`-OH groups (Table 3,
experiment 2). The decoding center at the P site, which is involved in
the selection of the initiator tRNA, evidently does not recognize the
2`-OH groups at the P-site codon, but appears to be sensitive to the
lack of these groups at the E-site codon. However, tRNA binding to
the A site requires the presence of the 2`-OH groups of the A-site
codons (Table 2, experiment 3), since the lack of the 2`-OH
groups causes a drop in the tRNA-binding affinity by an order of
magnitude (Table 4). A-site binding is also strongly reduced if a
deoxyribo-codon is present at the adjacent P site (Table 3,
experiment 3). The decoding center at the A site, where the decoding of
the genetic message during the elongation cycle takes place, is
therefore recognizing 2`-OH groups of codons at both A and P sites.
tRNA binding to both A and P sites is thus impaired if respective
upstream codons are lacking the 2`-OH groups. This observation reveals
a new kind of codon-context effect during translation and supports the
idea (24) that the decoding center of the elongating ribosome
interacts with the sugar-phosphate backbone of two adjacent codons
either in the A and P sites (pretranslocational complex) or in the P
and E sites (post-translocational complex). Decoding at the P site
(probably related to the initiation process) does not take into account
the 2`-OH groups of the corresponding codon but does so at the A site.
The recognition pattern at the P site therefore seems to be simpler
than that at the A site. This is not unexpected, since the
discrimination constraints weighed on codon-anticodon interaction at
the P site are relaxed compared with those at the A site. During
initiation the discrimination energy is not solely provided by
codon-anticodon interaction, but the initiation factors IF-2 and IF-3 (25) contribute as well as some unique features of the
initiator-tRNA such as the three G-C pairs closing the anticodon
loop(26) . It has indeed been shown that the
codon-anticodon-dependent accuracy for Ac-aminoacyl-tRNA binding is
significantly lower at the P site than at the A site (about 6-fold
lower under the conditions applied)(27) . During elongation
the discrimination problem is significantly more serious at the A site,
since (a) discrimination is restricted to codon-anticodon
interaction and (b) in E. coli, for example, one out
of 41 different tRNAs has to be selected (here tRNAs are considered as
different when they differ in their anticodon)(28) . The
problem is aggravated by the fact that it is not aminoacyl-tRNA
molecules which are selected but rather ternary complexes, where EF-Tu
accounts for 2/3 of the total mass (about 70 kDa) and increases the
affinity to the A site by more than 30-fold (19) . However,
EF-Tu adds nothing to the discrimination energy, since it is identical
in all ternary complexes. Considering on one hand the problem of
selection and, on the other hand, the accuracy of translation being
usually better than 1:1000 (one wrong amino acid incorporated per 1000
amino acids), it is clear that the discrimination energy provided by
the lifetimes of base pairing during codon-anticodon interaction is not
sufficient. The latter allows an accuracy of only 1:10 up to
1:100(1) . As outlined in the Introduction, this dilemma can be
solved in two ways. (a) The discrimination energy derived from
the stabilities of base pairing has to be used repeatedly, an idea
which has provoked the proofreading concepts for
translation(4, 5, 29, 30) . (b) The discrimination energy is larger than previously
assumed, since the nonaqueous conditions possibly established by the
decoding center may strengthen the energetics of interactions, and
other parameters than the lifetimes of codon-anticodon interactions are
exploited for the recognition process. One possible concept has been
proposed by Potapov (6) , i.e. the stereochemistry of
a correct partial structure of a Watson-Crick duplex formed by
codon-anticodon interactions is recognized by the decoding center at
the A sites. Correctly positioned sugars and phosphate groups are
contact sites of the decoding center and thus contribute to the
discrimination energy. A comparison of the effects of ribo-codons versus 2`-deoxyribo-codons represents a decisive experiment,
since an RNA-DNA hybrid forms an overall A-form structure as does an
RNA-RNA duplex, but the sugar pucker of the DNA strand is significantly
distorted in the hybrid(7) . Concept (a) should be
insensitive to the lack of 2`-OH groups in the A-site codons, whereas
concept (b) should be sensitive. Our results give a clear-cut
answer. The sugar structure is recognized at the A-site decoding center
during tRNA binding. This observation provides strong evidence that the
partial Watson-Crick structure is bound and recognized, whereas
incorrect duplex structures are not stably bound. It readily explains
why the accuracy is almost independent of the base pair character, i.e. whether or not a codon is rich in A/Us or G/Cs. It is
clear that the lifetime concept is not an appropriate model for
decoding. Since the recognition of Watson-Crick structures allows many
ribosome- tRNA contacts and thus large discrimination energies, one has
to envisage the possibility that the accuracy of translation is
achieved with no need of proofreading mechanisms. The well documented
fact that misincorporation of an amino acid is accompanied by an
increased GTPase turnover has been taken as the crucial argument in
favor of the existence of proofreading mechanisms in tRNA selection by
ribosomes. However, alternative scenarios have been described which
explain the excess of GTP cleavages during the selection of
near-cognate aminoacyl-tRNA without assuming proofreading mechanisms (31) ; furthermore, the excess of GTP cleavages is much lower
than previously assumed(32, 33) . Transcriptases
probably also recognize Watson-Crick structures during RNA
synthesis(34) , and they provide an example where high accuracy
is achieved (better than 1:50,000) (35) without proofreading.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-GAA-UUC-GUC-C
coding for glutamic acid (E),
phenylalanine (F) and valine (V), respectively, in the middle
(EFV-mRNA). A second template, the E(dF)V-mRNA, was identical except
that it carried a deoxyribo-codon -dUdUdC- for phenylalanine. tRNA
binding to the P site is totally insensitive to the presence or absence
of the 2`-OH group of the P-site codon, and tRNA binding to the P site
is also not affected if the A-site codon lacks the 2`-OH groups.
However, binding is impaired if the deoxy-codon is present at the E
site. In sharp contrast, the A-site binding of Ac-aminoacyl-tRNA was
severely reduced in the presence of the deoxy-codon at the A site as
well as at the P site. The results demonstrate that the correctness of
base pairing is also ``sensed'' via a correct sugar structure
of the codon, e.g. positioning of the sugar pucker (2`-OH),
during the decoding process at the A site (elongation) but not during
the decoding at the P site (initiation).
association constant for the deoxyribo-triplet p-dTdTdT is not
less, but even slightly higher than that for the ribo-triplet
p-UUU(14) . Therefore, the absence of translational activity of
poly(dT) cannot be explained by the weakness of the corresponding
codon-anticodon interactions per se. Specifically the P site
was tested by Ricker and Kaji(15) , who reported that deoxy
triplets p-dAdUdG or dAdTdG were as efficient as ribo-AUG in directing
the nonenzymatic binding of fMet-tRNA to the P site.
, tRNA
, and
tRNA
(E. coli) were purchased from
Subriden RNA, Rollingbay, WA. tRNA
(E. coli) was
from Boehringer Mannheim. All radioactive amino acids and
[
-
P]ATP were from Amersham Corp., HEPES was
from Calbiochem, and polyamines were from Fluka. Reversed phase columns
Nucleosil 300-5 C
and Nucleosil 300-7 C
were from Macherey-Nagel, Dren, FRG. All
other chemicals were from Merck, Darmstadt, and Boehringer Mannheim,
FRG.
,
Met
), as described elsewhere(16) . 1 A
unit of 70 S ribosomes was taken to be
equivalent to 24 pmol, 1 A
unit of tRNA to 1500
pmol.
Synthesis and Purification of mRNAs
Ribo-templates
(CGAAUUCGUCC
and
C
AUGGUCUUCC
) and ribo/deoxyribo-templates
(C
GAAdUdUdCGUCC
and
C
AUGGUC-dUdUdCC
) were synthesized with
dimethoxytrityl-cyanoethyl RNA/DNA phosphoramidites on an Applied
Biosystems 392 DNA-RNA synthesizer. Deprotected mRNAs were purified by
preparative electrophoresis in 17.5 or 20% PAAG with 7.5 M urea. Ribo- and ribo/deoxyribo-variants of the templates were
prepared and analyzed in parallel. 1 A
unit of
the templates was taken to be equivalent to 3000 pmol.
Aminoacylation and Purification of
tRNA
tRNA and tRNA
were
aminoacylated, and Phe-tRNA and Val-tRNA were acetylated as previously
described(16) . Phe-tRNA, Val-tRNA, AcPhe-tRNA, and AcVal-tRNA
were purified by reversed phase high performance liquid chromatography
on Nucleosil 300-5 C
and Nucleosil 300-7
C
columns using a linear (0-30%) or step (60%)
gradient of methanol in 400 mM NaCl, 10 mM
Mg(acetate)
, and 20 mM NH
(acetate), pH
5.0. After the purification, charging levels of 1600-1700
pmol/A
were obtained. AcPhe-tRNA and AcVal-tRNA
were made free from the corresponding aa
-tRNAs (
)by preparative deacylation of aa-tRNAs remaining in the
Ac-aa-tRNA fractions. For this purpose, Ac-aa-tRNA fractions were
treated with tRNA-free S-100 fraction used for aminoacylation. The
treatment was done in the absence of ATP and the cognate amino acid but
in the presence of AMP and pyrophosphate, both at a final concentration
of 6 mM.
Binding of tRNAs to Ribosomes
Binding of tRNAs and
Ac-aa-tRNAs to ribosomes was performed as described
elsewhere(16) . Analysis was done in a buffer system containing
20 mM HEPES-KOH (pH 7.5), 6 mM MgCl, 150
mM NH
Cl, 4 mM 2-mercaptoethanol, 0.05
mM spermine, and 2 mM spermidine. The standard assay
(final volume of 25 µl) contained about 10 pmol of 70 S ribosomes,
not less than 10-fold molar excess of a corresponding template, and
different combinations of tRNAs. Binding of tRNAs to ribosomes was
performed in two steps. During the first step, ribosomes and templates
were incubated at 37 °C in the absence or presence of deacylated
tRNA to prefill the ribosomal P site. During the second step, the
preformed complex was incubated at 37 °C with added
Ac-aminoacyl-tRNA. The presence of Ac-aminoacyl-tRNAs at the A or P
sites was tested with the puromycin reaction, which was carried out at
0 °C for 15-16 h as described elsewhere(16) . Before
adding, the pH of the freshly prepared puromycin solution was adjusted
to 7.5.
-three codons-C
with a total length of 41 nucleotides, which is about the length
of an mRNA protected by the ribosome against RNase
attack(17, 18) . Each of the three codons appears only
once in the mRNA sequence. One pair of mRNA molecules codes for
glutamic acid (E), phenylalanine (F), and valine (V), whereby the
EFV-mRNA contains the Phe codon in the usual ribo-form UUC and the
E(dF)V-mRNA in the 2`-deoxy form dUdUdC. The second pair of mRNAs, the
MVF-mRNA and the MV(dF)-mRNA, also carry a ribo-codon and
deoxyribo-codon for Phe, respectively, but in a different context.
(AcPhe-tRNA) to the Phe-codon
at the P site or the A site (Table 2). In a control experiment we
assess the binding to nonprogrammed 70 S ribosomes, which occurs
exclusively at the P site as indicated by the puromycin reaction (see
also (19) and references therein). The binding of AcPhe-tRNA
to programmed P sites is increased by about 25% compared with the
nonprogrammed P site (experiment 2, compare 2.9 and 3.0 with 2.2 pmol).
The interesting point is that the reactions depending on the ribo-codon
or on the deoxyribo-codon of Phe are indistinguishable, i.e. the P site is completely insensitive to the presence or absence of
2`-OH groups in its codon.
(codon GAA) can
efficiently bind to the ribo-codon in the P site, thus displaying the
deoxyribo-codon at the A site. A control experiment with deacylated
[
P]tRNA confirmed that the P site can be equally
well saturated whether or not a deoxyribo-codon is at the adjacent A
site (data not shown).
(20) . Other factors might also
contribute to the low binding, e.g. a higher sensitivity of
some nucleotides of the Val-tRNA
against the acetylation
procedure of the
-amino group of valine compared with
Phe-tRNA
. Since qualitatively the same results were
obtained with tRNA
we show here the results with
tRNA
in order to demonstrate that the observed effects do
not depend on the tRNA species used or on the codon present but rather
on the decoding process at the site under observation.
, AcPhe-tRNA binds to the P sites, whereas in the
presence of tRNA
it binds to the A sites. The binding
values were processed according to Scatchard (21) (Fig. 1, B and D), which gives
the corresponding association constants. These data are summarized in Table 4. The binding to the P sites occurs with the same affinity
regardless of whether a ribo-codon or a deoxyribo-codon is present at
the P site in agreement with the qualitative data shown in Table 2, experiment 2, with the EFV/E(dF)V pair of mRNAs. The
saturation curves also reveal an A-site affinity (ribo-codon), which is
twice as high as the corresponding P-site affinity (11
10
and 5.1
10
M
,
respectively). However, the affinity at the A site drops 10 times (from
11
10
to 1.1
10
M
) if a deoxyribo-codon is displayed
at the A site, in qualitative agreement with Table 2, experiment
3.
is
the number of bound AcPhe-tRNA molecules per ribosome, c is
the concentration of free AcPhe-tRNA. One assay (25 µl) contained
7.4 pmol of 70 S ribosome and either 157 pmol of MVF (A, B) or 154 pmol of MV(dF) (C, D). After a
preincubation for 30 min at 37 °C in the absence or the presence of
50 pmol of tRNA
the indicated amounts of
Ac[
C]Phe-tRNA (1054 dpm/pmol) were added, and
the systems were incubated for 60 min at 37
°C.
We thank Dr. R. Brimacombe for discussions and are
indebted to B. Rhrdanz and D. Kamp for their help.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.