© 2005 Nature Publishing Group http://www.nature.com/nsmb
ARTICLES
Idiosyncratic tuning of tRNAs to achieve uniform
ribosome binding
Mikołaj Olejniczak1,2, Taraka Dale1, Richard P Fahlman1,3 & Olke C Uhlenbeck1
The binding of seven tRNA anticodons to their complementary codons on Escherichia coli ribosomes was substantially impaired,
as compared with the binding of their natural tRNAs, when they were transplanted into tRNAAla
2 . An analysis of chimeras
Gly
Arg
composed of tRNAAla
2 and various amounts of either tRNA3 or tRNA2 indicates that the presence of the parental 32-38
nucleotide pair is sufficient to restore ribosome binding of the transplanted anticodons. Furthermore, mutagenesis of tRNAAla
2
showed that its highly conserved A32-U38 pair serves to weaken ribosome affinity. We propose that this negative binding
determinant is used to offset the very tight codon-anticodon interaction of tRNAAla
2 . This suggests that each tRNA sequence has
coevolved with its anticodon to tune ribosome affinity to a value that is the same for all tRNAs.
Although aminoacylated transfer RNAs (aa-tRNAs) are deeply
embedded within the ribosomal A and P sites during translation1,
only a few of the contacts between tRNA and the ribosome involve
tRNA bases2,3. Structural data show that the A and P sites form
multiple contacts with 2¢-OH groups and phosphates in the acceptor
and anticodon stems of the tRNA, as well as with the folded tRNA
core1,4. The substitution of several of these functional groups results in
weaker tRNA binding5,6 or altered function7,8. This design of the
ribosomal A and P sites presumably permits the ribosome to accommodate the many tRNA bodies interchangeably. Indeed, recent
biochemical experiments have established that different elongator
aa-tRNA species show remarkably uniform binding affinities for the
ribosomal A and P sites9. Although this result is consistent with their
interchangeable roles in translation, an important challenge is to
understand how a diverse set of tRNA bodies can interact with
ribosomes in a way that yields similar affinities. Notably, when the
esterified amino acids are removed from each aminoacyl-tRNA, tRNA
binding to the A and P sites is no longer uniform9. This observation
suggests that both ribosomal sites show specificity for tRNA bodies
and that each body has evolved to compensate for differences in the
contributions of the side chains of the esterified amino acids to the
tRNA’s affinity for the ribosome.
It is likely that the different tRNA bodies have also evolved to
compensate for intrinsic differences in the strengths of anticodoncodon interactions. The affinity of a tRNA for ribosomes is dominated
by the interaction of the anticodon stem-loop of tRNA with the 30S
subunit10 containing the codon. Although the anticodon-codon base
pairs are extensively stabilized by buttressing tertiary interactions
provided by the small ribosomal subunit11,12, it is unclear whether
these interactions are sufficient to homogenize the large intrinsic
differences in the strengths of anticodon-codon interactions dictated
by the G+C content. Other ribosomal contacts with the tRNA body
may therefore be used to adjust the affinities of different tRNA
sequences. Indeed, numerous studies have identified elements outside
the anticodon that modulate tRNA performance, possibly through
A- or P-site binding. These include post-transcriptional tRNA
modifications, especially at the neighboring position 37 (ref. 13), as
well as sequence elements in the anticodon stem-loop14–16 and elsewhere in the tRNA body17.
To understand how sequence differences among tRNA bodies could
contribute to ribosome binding, we compared the stabilities of seven
different anticodons embedded either in their natural tRNAs or in the
foreign body of tRNAAla
2 when these anticodons were bound to their
complementary codons in the P and A sites of E. coli ribosomes.
RESULTS
Ribosome binding of tRNAs with transplanted anticodons
Seven anticodons with a range of anticodon-codon sequences were
tested for their ability to bind ribosomes when embedded in their
natural tRNA sequences and when transplanted into tRNAAla
2 . The
seven natural tRNAs were the same as we had studied previously9.
tRNAAla
2 was chosen to be the recipient tRNA for the anticodon
transplants because its few post-transcriptional modifications and
esterified alanine contribute little to ribosome binding under the
assay conditions (data not shown). Therefore, unmodified, deacylated
tRNAs could be used in these experiments.
The ACG anticodon binds its complementary CGU codon much
better in the context of its parental tRNAArg
sequence than when
2
transplanted into tRNAAla
2 . The stabilities of the two tRNAs (Fig. 1a)
in both the ribosomal P site and A site were initially assessed by
1Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, USA. 2Institute of
Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznań, Poland. 3Present address: Centre for Blood Research, University of British
Columbia, Vancouver, British Columbia V6T 1Z3, Canada. Correspondence should be addressed to O.C.U. ([email protected]).
Published online 21 August 2005; doi:10.1038/nsmb978
788
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b
A
C
C
A
GC
CG
A U
UA
CG
CG
GC
A
U
C C UC CU A
A
GA
C
G G A GGU CG
U
CUCG
U
CU
G
U
G
G
A
GA U A
G
A
C GC G
UA
CG
GC
GC
C A
A
U
A CG
Figure 1 The ACG anticodon transplanted into tRNAAla
2 binds poorly to
Ala
E. coli 70S ribosomes. (a) Sequences of tRNAArg
2 (left) and tRNA2 (ACG)
(right). (b) Rates of dissociation from the A site (left) and P site (right) for
Arg
Ala
tRNAAla
2 , tRNA2 and tRNA2 (ACG). koff values are given in Tables 1 and 2.
Arg
(c) Equilibrium binding to A site (left) and P site (right) for tRNAAla
2 , tRNA2
Ala
and tRNAAla
2 (ACG). Binding of tRNA2 (ACG) to the A site in the presence
Ala
of paromomycin is also shown (left). Kd values for tRNAAla
2 and tRNA2
(ACG) are given in Tables 1 and 2. tRNAArg
2 has a Kd of 150 nM for the
A site and 20 nM for the P site. The binding of tRNAAla
2 (ACG) to the A site
in the presence of 100 mM paromomycin has an apparent Kd ¼ 36 nM.
A
C
C
A
GC
GC
GU
GC
CG
UA
AU
U
U
U CG C C C A
A
C GA
A G CGGU CG
U
CUCG
U
CU
G
C
G
G
A
GG A
G
G
C G AG
UA
UA
GC
CG
A U
A
U
A CG
measuring their dissociation rates using a chase protocol described
previously18. Although natural tRNAAla
(anticodon GGC) and
2
tRNAArg
2 (ACG) bind their cognate codons quite well, the dissociation
rate of tRNAAla
(ACG) binding complementary codons in both
2
the P and A sites is too fast to accurately measure using manual
pipetting methods (Fig. 1b). This result indicates that the tRNAAla
2
sequence context is detrimental for binding of the ACG anticodon to
its cognate codon.
The poor binding of tRNAAla
2 (ACG) to ribosomes was confirmed
using an assay that determines Kd directly. In this assay, a trace
concentration of 3¢ 32P-labeled tRNA was incubated with various
concentrations of ribosomes in the presence of mRNA, either with a
saturating concentration of tRNAfMet to measure A-site binding or
without it to measure P-site binding. tRNAArg
and tRNAAla
2
2 bound
cognate codons quite tightly in both the P and A sites (Kd B20 nM
and B200 nM, respectively; Fig. 1c). In contrast, tRNAAla
2 with the
transplanted ACG anticodon bound the P site approximately 20-fold
more weakly than wild-type tRNAAla
2 , and its A-site affinity was too
weak to obtain an accurate Kd. The poor A-site binding was remedied
by the addition of 100 mM of paromomycin (Fig. 1c), an antibiotic
that stabilizes tRNA binding to the A site19. Thus, the poor binding of
tRNAAla
2 (ACG) was specifically a consequence of replacing the anticodon and not the result of misfolding of the tRNA such that it could
not bind ribosomes.
The ribosome-binding properties of the other six anticodons were
also determined in both the natural and tRNAAla
contexts. The
2
dissociation rates of the eight natural tRNAs (including tRNAAla
2 )
from their respective complementary codons were substantially different, with koff values ranging between 6.3 and 120 103 min–1 in
the A site (Table 1) and between 6.1 and 440 103 min–1 in the
P site (Table 2). As the fully modified, aminoacylated tRNAs all
dissociate from the A site and the P site at similar rates9, the
differences among dissociation rates are due to a combination of
the absence of modifications and of esterified amino acids. We
emphasize, however, that each anticodon bound the ribosomal A
and P sites substantially better when it was embedded in its natural
tRNA than when it was transplanted into tRNAAla
2 . For example,
transplantation of the GCC anticodon from its natural tRNAGly
3 into
tRNAAla
2 resulted in about 15-fold weaker binding in the A site and
ten-fold weaker binding in the P site. In the cases of tRNAAla
2 with the
GUA, GUG, ACG or UUC anticodons, the effect was even more
marked. These tRNAs dissociated from either site too rapidly to
measure the koff, although weak Kd values in the P site (but not the
A site) could be obtained. In each case, very weak A-site binding could
be restored by the addition of paromomycin, confirming that none
Table 1 A-site binding of anticodons in different tRNA contexts
Table 2 P-site binding of anticodons in different tRNA contexts
0
0
tRNA Ala
2
–2
tRNA Arg
2
–3
–4
–5
ln (fraction bound)
ln (fraction bound)
–1
tRNAAla
2
–1
tRNAArg
2
–2
–3
tRNAAla
2 (ACG)
tRNA Ala
2 (ACG)
–4
0
5
10
15
20
Time (min)
25
30
0
10
20
30
Time (min)
40
50
c 0.5
tRNAAla
2
tRNAAla
2 (ACG)
(paromomycin)
tRNAArg
2
0.3
0.2
tRNAAla
2 (ACG)
0.1
tRNAArg
2
0.6
0.4
1
10
100
1,000
Ribosome concentration (µM)
tRNAAla
2
(ACG)
0.2
0
0
tRNAAla
2
0.8
Fraction bound
0.4
Fraction bound
© 2005 Nature Publishing Group http://www.nature.com/nsmb
a
1
10
100
1,000
Ribosome concentration (µM)
Anticodon in natural
tRNA sequence
Anticodon in natural
Anticodon in tRNAAla
2 sequence
Anticodon
tRNA sequence
Anticodon in tRNAAla
2 sequence
Anticodon
(natural tRNA)
koff (min–1 103)
koff (min–1 103)
Kd (nM)
(natural tRNA)
GGC (tRNAAla
2 )
32 ± 7
32 ± 7
170 ± 81
GGC (tRNAAla
2 )
12 ± 3
12 ± 3
20 ± 10
ACG (tRNAArg
2 )
UUC (tRNAGlu)
52 ± 19
110 ± 28
4500
4500
41,000
41,000
ACG (tRNAArg
2 )
UUC (tRNAGlu)
15 ± 3
55 ± 16
4500
4500
550 ± 160
610 ± 150
GCC (tRNAGly
3 )
6.3 ± 2.4
94 ± 33
240 ± 55
GCC (tRNAGly
3 )
6.1 ± 1.6
67 ± 16
GUG (tRNAHis)
GAA (tRNAPhe)
95 ± 22
120 ± 40
4500
4500
41,000
41,000
GUG (tRNAHis)
GAA (tRNAPhe)
440 ± 130
120 ± 40
4500
260 ± 76
590 ± 100
270 ± 99
GUA (tRNATyr)
GAC (tRNAVal
2a )
19 ± 12
13 ± 4
4500
30 ± 6
41,000
220 ± 52
GUA (tRNATyr)
GAC (tRNAVal
2a )
130 ± 28
13 ± 4
4500
40 ± 6
820 ± 140
22 ± 10
NATURE STRUCTURAL & MOLECULAR BIOLOGY
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NUMBER 9
koff (min–1 103)
SEPTEMBER 2005
koff (min–1 103)
Kd (nM)
59 ± 9
789
A site
P site
32 ± 7
12 ± 3
94 ± 33
67 ± 16
9.1 ± 1.9
8.4 ± 2.2
10 ± 2
6.7 ± 1.1
11 ± 2
8.1 ± 1.8
6.3 ± 2.4
6.1 ± 1.6
A site
P site
32 ± 7
12 ± 3
>500
>500
51 ± 12
25 ± 5
62 ± 20
14 ± 5
71 ± 18
18 ± 5
52 ± 19
15 ± 3
Gly
Arg
Figure 2 Chimeric tRNAs combining tRNAAla
2 (black) with tRNA3 (red, upper row) or tRNA2 (green, lower row). Dissociation rates from A and P sites are
indicated (min–1 103).
of the tRNAs were misfolded. These experiments show that the
anticodons function much better in their natural setting than when
transplanted into the foreign tRNAAla
2 body.
Dissection shows the importance of the 32-38 pair
To understand the reason for the incompatibility of the tRNAAla
2 body
with the transplanted anticodons, a series of chimeric tRNAs were
prepared that introduced increasing portions of a donor tRNA
Ala (GCC)
sequence into tRNAAla
2 . This was done both for tRNA2
with tRNAGly
,
where
the
deleterious
effect
of
the
anticodon
transplant
3
was relatively modest, and for tRNAAla
(ACG) with tRNAArg
2
2 ,
where the transplant markedly reduced binding (Fig. 2). As the
Gly and tRNAArg are similar
overall structures of tRNAAla
2 , tRNA3
2
and the design of the chimeras was conservative, it seemed likely
Gly
that the chimeras would fold normally. In the tRNAAla
2 -tRNA3
chimeras, the introduction of the U32-A38 pair from tRNAGly
3
into the tRNAAla
2 context was sufficient to restore the koff to that of
the natural tRNAGly
3 . Introduction of the whole anticodon stem-loop
region or both the anticodon stem-loop and core regions of
tRNAGly
into tRNAAla
3
2 did not further enhance binding. A similar
Arg chimeras, where the
result was obtained for tRNAAla
2 -tRNA2
introduction of the C32-A38 pair from tRNAArg
into tRNAAla
2
2
Arg
was sufficient to restore the koff to that of tRNA2 . Neither the
introduction of the G31-C39 pair nor that of the whole anticodon
stem of tRNAArg
increased the binding any further. These results
2
suggest that the A32-U38 pair in tRNAAla
2 is the major reason for the
observed incompatibility with foreign anticodons. When this pair is
converted from A-U to the U-A found in tRNAGly
3 or C-A found in
tRNAArg
2 , the ribosome binding activity is restored. In other words, the
A32-U38 pair acts as a negative determinant in the anticodontransplanted tRNAs.
Tuning of tRNAAla
2 for ribosome binding
To test whether the A32-U38 pair could also act as a negative
determinant for ribosome binding of the natural GGC anticodon
present in tRNAAla
2 , a set of constructs containing mutations at these
790
VOLUME 12
positions in the tRNAAla
2 background were assayed. As A32-U38 is
rarely seen in other tRNAs20, including tRNAAla
1 , the pair was changed
to U32 . U38, A32 . A38 or U32-A38, which are present in many
tRNAs. tRNAs with these mutations dissociate from the ribosomal A
site four- to ten-fold more slowly than wild-type tRNAAla
2 (Fig. 3).
About two-fold slower dissociation is observed in the P site as well
(Fig. 4). Thus, even when its natural GGC anticodon is present, the
A32-U38 base pair weakens the binding of E. coli tRNAAla
2 to the
ribosome. Notably, a comparison of nonredundant tRNAAla
2 isoacceptor gene sequences in the tDNA database21 shows that the A32-U38
pair is present in 42 of 56 bacterial species and that the remainder
contain the very rare C32-G38 pair. When the C32-G38 pair was
introduced into E. coli tRNAAla
2 , the tRNA bound the ribosome
similarly to the wild-type A32-U38 tRNAAla
2 . Thus, the weakened
affinity for ribosomes caused by the A32-U38 or C32-G38 pair is a
phylogenetically conserved feature of tRNAAla
2 isoacceptors that has
coevolved specifically with the GGC anticodon.
0
U32-A38
A32•A38
U32•U38
–0.5
In (fraction bound)
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ARTICLES
–1
–1.5
C32-G38
–2
A32-U38
–2.5
0
10
20
30
Time (min)
40
Figure 3 Rate of dissociation of tRNAAla
2 with different 32-38 pairs from
the A site. Lines are best-fit dissociation rates of tRNAAla
2 with: A32-U38
(wild type), koff ¼ 31 103 min–1; C32-G38, koff ¼ 26 103 min–1;
U32 . U38, koff ¼ 5.4 103 min–1; A32 . A38, koff ¼ 4.7 103 min–1;
U32-A38, koff ¼ 2.8 103 min–1.
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© 2005 Nature Publishing Group http://www.nature.com/nsmb
32 ± 7
tRNAAla
2
12 ± 3
39 ± 7
18 ± 5
C-G
31 ± 4
49 ± 15
U-A
36 ± 5
60 ± 10
A-U
29 ± 9
14 ± 5
U-A
27 ± 9
7.7 ± 0.9
G-C
C27 – G
U28 – A
U29 – A
G30 – C
C31 – G
A32
U38
U
A
GGC
A-U
23 ± 4
9.7 ± 2
C-G
C-G
33 ± 7
27 ± 6
U-A
3.1 ± 0.7
6.4 ± 1.7
U•U
7.3 ± 1.9
4.8 ± 1.6
A•A
6.5 ± 2.1
5 ± 1.7
C-G
24 ± 2
10 ± 2
Figure 4 Ribosome binding determinants in the tRNAAla
2 body. Next to each
base-pair mutation, koff values (min–1 103) for A site (above) and P site
–1 103).
(below) are shown. Top, wild-type tRNAAla
2 koff values (min
As X-ray crystal structures of the 70S ribosome1 and the 30S
subunit11 show specific contacts between the 16S rRNA and the
anticodon stem in both the A and P sites, we wished to test whether
the nucleotides in the anticodon stem might also affect tRNAAla
2
binding to the ribosome. None of the mutations of tRNAAla
2 that
changed the base pairs in the anticodon stem affected koff at the A site
(Fig. 4). However, mutants containing changes at the 30-40 base pair
had a substantially faster rate of dissociation from the P site. When the
natural G30-C40 pair was replaced with C30-G40, the effect was small;
however, with both A30-U40 and U30-A40, dissociation was more
than four-fold faster. Slightly faster rates of dissociation from the P site
were also observed for the U28C-A42G, U29C-A41G mutant. In
agreement with these observations, the nucleotides in positions 30,
40 and 41 have been shown to interact with the G1338 and
A1339 nucleotides of 16S rRNA1,4, which are important for P-site
binding22–24. Notably, the identity of the nucleotide in position 40 is
also important for frameshifting25,26, and this effect could be
explained as a result of changes in tRNA interactions with the
P site27. We were surprised to find that base-pair mutations at
positions 31-39 and 27-43 had no effect on the dissociation of tRNAAla
2
from the A site or P site even though the identities of these nucleotides
are known to be important for the function of other tRNAs15,16,28. In
any case, it is evident that the tRNAAla
2 body contains both negative
and positive sequence determinants, which tune its binding affinity in
a way that is specific for each ribosomal site.
DISCUSSION
Most of the tRNAAla
molecules with foreign anticodons that we
2
constructed bound ribosomes poorly and thus would not be expected
to function well in translation. Although the anticodons of many
tRNAs can be mutated so that they function as suppressor tRNAs,
achieving sufficient suppression efficiency often requires extensive
mutations in the tRNA body14,16. The poor performance of tRNAAla
2
with transplanted anticodons was traced to the rare A32-U38 pair.
When this pair was converted to one of several other 32-38 combinations, the binding affinities of chimeric tRNAs increased substantially.
These results are consistent with the observation that the inefficient
tRNAAla
2 (CUA) amber suppressor can be improved by the mutation
U38A29, which presumably causes tighter binding and greater A-site
occupancy. There is abundant evidence from previous research on
suppressor tRNAs that other 32-38 pairs can contribute to tRNA
function in a way that is somewhat specific to a given tRNA body.
NATURE STRUCTURAL & MOLECULAR BIOLOGY
VOLUME 12
For example, the efficiency of the tRNAAla
1 -derived suppressor
depends on the 32-38 residues, which increasingly promote suppres30
sion in the order UU o UC (natural for tRNAAla
1 ) o CC o CA ,
whereas both the tRNATrp- and tRNAGln-derived amber suppressors
show efficiency varying with 32-38 base pairs in the order Um-C o
Um-C r Um-A o Cm-A31 Um, 2¢-O-methyluridine; Cm, 2¢-Omethylcytidine; C, pseudouridine. Although much less data is available, there is evidence that mutation of the 32-38 pair can affect the
translation efficiency of normal elongator tRNAs as well. For example,
the U32C mutation in tRNAGly
1 weakens cognate codon recognition
in vitro32,33 and increases frameshifting efficiency in vivo34. Thus, the
32-38 pair contributes substantially to the translational function of
many tRNAs.
On the basis of available X-ray crystal structures of tRNAs and
tRNA-protein complexes, the majority of 32-38 pairs have been
assigned to two families of isosteric, non–Watson-Crick base pairs
that maintain the structure of the anticodon loop20. Notably, the rare
A32-U38 pair predominantly present in tRNAAla
2 isoacceptors is not
isosteric to either common structural family of 32-38 pairs20,35. The
phylogenetic analysis shows that all of the tRNAAla
2 molecules without
A32-U38 contain the very rare C32-G38 pair, suggesting that 32-38
nucleotides in tRNAAla
2 form a Watson-Crick base pair. As A37 in
tRNAAla
2 is not modified, a Watson-Crick pair could also form at U33A37, resulting in a triloop conformation, which is detrimental for the
ribosome binding of other tRNAs36–38. Anticodon sequence could also
influence loop conformation, which may lead to conformational
differences among the anticodon loops of tRNAAla
2 (GGC) and its
anticodon-swapped derivatives, thus explaining differences in their
affinities for the ribosome. In agreement with that suggestion, hairpins
containing GGC triloop sequence are less stable than those containing
uridine-rich sequences39. Thus, an altered anticodon-loop conformation could result in suboptimal tRNA contacts with the ribosome. This
would explain the weaker A-site binding of wild-type tRNAAla
2 in
comparison to mutants with more common 32-38 combinations that
are known to permit a normal anticodon-loop structure20,35.
Regardless of the structural explanation, the presence of the
natural A32-U38 pair results in weaker binding of E. coli tRNAAla
2 to
its cognate codon. As this pair is very strongly phylogenetically
conserved in tRNAAla
2 , it appears that there is strong selective
pressure to ‘tune’ tRNAAla
2 to bind the A-site less efficiently than it
could. What could be the reason for such a negative determinant?
One possibility is that the tRNAAla
2 GGC anticodon is capable of
forming an exceptionally stable complex with its GCC codon in the
A site. Not only does the anticodon-codon complex consist of three
stable G-C pairs, but the two buttressing A-minor interactions at
the first and second codon positions are more effective at stabilizing
C-G or G-C pairs than U-A pairs40. Too tight A-site binding
could deleteriously affect translation in many ways, including slowing
one of the crucial conformational changes during decoding41 or
translocation42, or promoting frameshifting by stabilizing out-offrame codons43. Thus, by having the deleterious A32-U38 pair,
E. coli tRNAAla
2 avoids binding ribosomes too tightly. It is noteworthy
that the only other bacterial tRNA isoacceptor that contains the
A32-U38 pair (in 35% of species) or the also potentially deleterious
C32-G38 pair (in 46% of species) is tRNAPro
2 (GGG), which is also
likely to have very stable anticodon-codon complex. In fact, many
anticodons are strongly phylogenetically correlated with a particular
type of 32-38 pair. For example, 96% of the GCC anticodons use the
U32-A38 pair, 99% of the ACG anticodons use C32-A38, 65% of the
GAC anticodons use U32 . U38 and 79% of the UUC anticodons use
C32-C38. This suggests that the 32-38 interaction may be universally
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ARTICLES
selected to compensate for inherent differences in anticodon-codon
binding strengths.
The idea that tRNA sequences are ‘tuned’ to optimize translation
efficiency is well established in suppressor tRNA literature. Yarus’s
‘extended anticodon’ hypothesis notes that residues on the 3¢ side of the
anticodon as well as several pairs in the anticodon stem are organized
predominantly to suit the properties of the ‘cardinal’ nucleotide at
position 36 of the anticodon and thereby enhance translational
efficiency44. Several experiments supporting this hypothesis show
that the suppression efficiency of a tRNA can be improved by changing
the sequence of the anticodon stem-loop so that it is appropriate to the
cardinal nucleotide14,16,31. Our discovery of an evolutionarily conserved
negative binding determinant in an elongator tRNA with a sense codon
clearly shows that not all tuning serves to improve the efficiency of a
tRNA. It seems that the role of this negative determinant is to weaken
the A-site (and possibly P-site) affinity of tRNAAla
2 to offset its very
tight anticodon-codon interaction. This in turn implies that one
evolutionary pressure on tRNA sequences is not to maximize ribosome
binding but to optimize it to a certain value. As eight other native
aa-tRNAs bind the ribosomal A and P sites with similar affinities9, it is
likely that the optimal value for Ala-tRNAAla
2 is the same as for all other
elongator tRNAs. Indeed, recent experiments measuring the affinity of
native Ala-tRNAAla
2 for the A site support this conclusion (T.D. and
O.C.U., unpublished data). Thus, it seems that there is a strong
selective pressure on tRNA sequences to achieve uniform ribosome
binding. It will be interesting to see whether other steps in the
translation mechanism are also uniform with different aa-tRNA substrates and whether they use different sequence elements for tuning.
The strategy by which aa-tRNAs achieve uniform ribosome binding
is expected to be different for each tRNA species and is dictated by
both the identity of the amino acid and the anticodon sequence. Those
differences are best illustrated by considering two very different
adaptations for E. coli tRNAs binding to the ribosome. tRNAs of
one group, typified by tRNAAla
2 (GGC), have a very stable anticodoncodon interaction because they are GC rich and have strongly
stabilizing A-minor interactions. Members of this group generally
lack post-transcriptional modifications or sequence elements in the
anticodon stem-loop that stabilize ribosome binding and may contain
negative sequence elements that weaken binding. In addition, the
esterified amino acid is not expected to contribute substantially to
ribosome binding for these tRNAs. Other members of this group
Gly
Val
probably include tRNAGly
3 (GCC), tRNA1 (CCC), tRNA2a (GAC)
and tRNAPro
(GGG),
which
all
have
stable
anticodon-codon
interac2
tions. Each uses a slightly different strategy combining modifications
and sequence elements to achieve uniform ribosome binding. tRNAPro
2
(GGG) contains the m1G37 modification as well as the negative A32U38 sequence determinant, whereas the others lack anticodon stemloop modifications and have U32-A38 or U32 . U38 pairs30,31. tRNAs
on the opposite end of the anticodon-codon stability spectrum contain
one or more uridines in the anticodon. Examples include tRNALys
(UUU), tRNAGlu (UUC) and tRNATyr (GUA). The U-A pairs formed
with their cognate codons not only are intrinsically weak but also are
expected to be stabilized less well than other pairs by the A-minor
interactions40. These tRNAs need anticodon stem-loop modifications
and positive sequence determinants, as well as the presence of the
amino acid, to compensate for their weak anticodons. As expected, all
of these tRNAs bind to ribosomes inefficiently in the absence of the
amino acid or their extensive modifications9. Notably, these tRNAs
contain C32-A38 or C32-C38 pairs, which are the most effective in
improving ribosomal performance of suppressor tRNAs30,31. Other
tRNAs, including the very well-characterized tRNAPhe (GAA), lie
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VOLUME 12
between these two extremes, and each one uses a unique combination
of sequence elements and modifications to tune ribosome binding.
Thus, it seems that the selective pressure to maintain a uniform affinity
for the ribosome is a driving force for the substantial diversity in tRNA
sequences and post-transcriptional modifications.
METHODS
Materials. Tightly coupled 70S ribosomes from E. coli MRE600 cells were
prepared as described in ref. 18. The mRNA fragments were purchased from
Dharmacon, deprotected according to producer’s protocol and purified by
denaturing PAGE. The mRNAs were derivatives of the initiation region of the
T4 gp32 mRNA that has been used for X-ray crystallographic studies of mRNA
bound to the ribosome45 and had the following sequence: 5¢-GGCAAGGAG
GUAAAAAUGXXXGCACGU-3¢, where XXX indicates the codon complementary to the anticodon of each analyzed tRNA.
Preparation and labeling of tRNAs. tRNAs were prepared by in vitro
Ala
Ala
transcription46. Transcription templates for tRNAVal
2a , tRNA2 and tRNA2
derivatives were generated by primer extension of overlapping DNA oligonucleotides (IDT). Transcription templates for other tRNAs were prepared by
PCR amplification of plasmid DNA. Transcribed tRNAs were purified on 10%
denaturing polyacrylamide gels.
The 3¢ 32P labeling was performed using [a-32P]ATP (Amersham) and tRNA
nucleotidyl transferase47.
tRNA dissociation from A and P sites. The kinetics of tRNA binding to the
ribosomes was determined by filter binding on a modified 96-well dot blot
apparatus (Schleicher and Schuell). An upper nitrocellulose membrane (Protran,
Schleicher and Schuell) and a lower nylon membrane (Hybond-N+, Amersham)
were soaked in ribosome binding (RB) buffer (50 mM HEPES (pH 7.0), 30 mM
KCl, 70 mM NH4Cl, 10 mM MgCl2 and 1 mM DTT) before use. Dissociation
rates were measured in RB buffer as described previously9,18 with the following
changes. Dissociation rates of anticodon-substituted tRNAs were initially
measured at 5 mM, 7.5 mM, 10 mM and 15 mM MgCl2 to confirm that in the
conditions of the experiment tRNAs are correctly bound to either the P site or
the A site. As seen previously (R.P.F., unpublished data), the dissociation rates of
tRNAs bound to the P site, but not the A site, are strongly dependent on Mg2+
concentration. Final measurements of the dissociation rates of all tRNAs were
performed at 10 mM MgCl2. Experiments measuring dissociation rates from the
P site usually used 0.3 mM ribosomes18; however, several of the weak tRNAs
required 1 mM ribosomes to ensure sufficient binding before the chase.
Equilibrium binding of anticodon-substituted tRNAs to A and P sites. Before
use, the ribosomes were activated at 42 1C for 10 min. For P-site binding
experiments, the stock ribosome solution contained RB buffer with 2 mM
ribosomes and 9 mM mRNA with a codon complementary to tRNA’s anticodon. For A-site binding experiments, the stock ribosome solution also
contained 10 mM tRNAfMet. For a single P-site binding experiment, a series
of two-fold serial dilutions were prepared by mixing 25 ml of stock with 25 ml of
0.6 mM mRNA in RB buffer and then removing 25 ml for the next dilution.
A-site binding experiments were performed similarly except that the dilution
buffer also contained 1.2 mM tRNAfMet. Fifteen microliters of each ribosome
dilution was mixed with 15 ml of 2 nM 3¢ 32P-labeled tRNA, resulting in a range
of ribosome concentrations from 1 nM to 1 mM. As the association rate of
tRNAs to ribosomes is slow, a 2-h incubation was required for tightly binding
tRNAs to reach equilibrium at lower ribosome concentrations. For weakly
binding tRNAs a 40-min incubation was sufficient. After incubation at 20 1C
for the appropriate time, the mixtures were filtered through the double filter
system and washed with RB buffer as described previously18. After drying the
filters, we quantified data using a phosphorimager (Molecular Dynamics) with
ImageQuant software. Equilibrium binding constants were obtained by fitting
to a sigmoidal binding curve using KaleidaGraph software (Synergy Software).
Phylogenetic analysis of 32-38 pair correlation with anticodon sequence. For
phylogenetic analysis, bacterial tRNA gene sequences from a genomic tRNA
database21 were selected. Duplicate tRNA sequences from the same organism
were not included unless they showed differences at positions 32, 38 or both.
NUMBER 9
SEPTEMBER 2005
NATURE STRUCTURAL & MOLECULAR BIOLOGY
ARTICLES
ACKNOWLEDGMENTS
This work was supported by US National Institutes of Health Grant GM 37552 to
O.C.U. We would also like to thank M. Saks for critically reading the manuscript.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
© 2005 Nature Publishing Group http://www.nature.com/nsmb
Received 1 June; accepted 22 July 2005
Published online at http://www.nature.com/nsmb/
1. Yusupov, M.M. et al. Crystal structure of the ribosome at 5.5 A resolution. Science 292,
883–896 (2001).
2. Samaha, R.R., Green, R. & Noller, H.F. A base pair between tRNA and 23S rRNA in the
peptidyl transferase centre of the ribosome. Nature 377, 309–314 (1995).
3. Kim, D.F. & Green, R. Base-pairing between 23S rRNA and tRNA in the ribosomal A
site. Mol. Cell 4, 859–864 (1999).
4. Carter, A.P. et al. Functional insights from the structure of the 30S ribosomal subunit
and its interactions with antibiotics. Nature 407, 340–348 (2000).
5. von Ahsen, U., Green, R., Schroeder, R. & Noller, H.F. Identification of 2¢-hydroxyl
groups required for interaction of a tRNA anticodon stem-loop region with the
ribosome. RNA 3, 49–56 (1997).
6. Schnitzer, W. & von Ahsen, U. Identification of specific Rp-phosphate oxygens in the
tRNA anticodon loop required for ribosomal P-site binding. Proc. Natl. Acad. Sci. USA
94, 12823–12828 (1997).
7. Feinberg, J.S. & Joseph, S. Identification of molecular interactions between P-site
tRNA and the ribosome essential for translocation. Proc. Natl. Acad. Sci. USA 98,
11120–11125 (2001).
8. Phelps, S.S., Jerinic, O. & Joseph, S. Universally conserved interactions between the
ribosome and the anticodon stem-loop of A site tRNA important for translocation. Mol.
Cell 10, 799–807 (2002).
9. Fahlman, R.P., Dale, T. & Uhlenbeck, O.C. Uniform binding of aminoacylated transfer
RNAs to the ribosomal A and P sites. Mol. Cell 16, 799–805 (2004).
10. Rose, S.J., III, Lowary, P.T. & Uhlenbeck, O.C. Binding of yeast tRNAPhe anticodon
arm to Escherichia coli 30 S ribosomes. J. Mol. Biol. 167, 103–117 (1983).
11. Ogle, J.M. et al. Recognition of cognate transfer RNA by the 30S ribosomal subunit.
Science 292, 897–902 (2001).
12. Sanbonmatsu, K.Y. & Joseph, S. Understanding discrimination by the ribosome:
stability testing and groove measurement of codon-anticodon pairs. J. Mol. Biol.
328, 33–47 (2003).
13. Agris, P.F. Decoding the genome: a modified view. Nucleic Acids Res. 32, 223–238
(2004).
14. Kleina, L.G. et al. Construction of Escherichia coli amber suppressor tRNA genes. II.
Synthesis of additional tRNA genes and improvement of suppressor efficiency. J. Mol.
Biol. 213, 705–717 (1990).
15. Komine, Y. & Inokuchi, H. Importance of the G27–A43 mismatch at the anticodon
stem of Escherichia coli tRNA(Thr2). FEBS Lett. 272, 55–57 (1990).
16. Raftery, L.A. & Yarus, M. Systematic alterations in the anticodon arm make tRNA(Glu)Suoc a more efficient suppressor. EMBO J. 6, 1499–1506 (1987).
17. Hirsh, D. Tryptophan transfer RNA as the UGA suppressor. J. Mol. Biol. 58, 439–458
(1971).
18. Fahlman, R.P. & Uhlenbeck, O.C. Contribution of the esterified amino acid to the
binding of aminoacylated tRNAs to the ribosomal P- and A-sites. Biochemistry 43,
7575–7583 (2004).
19. Ogle, J.M., Murphy, F.V., Tarry, M.J. & Ramakrishnan, V. Selection of tRNA by the ribosome requires a transition from an open to a closed form. Cell 111, 721–732 (2002).
20. Auffinger, P. & Westhof, E. Singly and bifurcated hydrogen-bonded base-pairs in tRNA
anticodon hairpins and ribozymes. J. Mol. Biol. 292, 467–483 (1999).
21. Sprinzl, M. et al. Compilation of tRNA sequences and sequences of tRNA genes.
Nucleic Acids Res. 26, 148–153 (1998).
22. Moazed, D. & Noller, H.F. Binding of tRNA to the ribosomal A and P sites protects two
distinct sets of nucleotides in 16 S rRNA. J. Mol. Biol. 211, 135–145 (1990).
NATURE STRUCTURAL & MOLECULAR BIOLOGY
VOLUME 12
23. von Ahsen, U. & Noller, H.F. Identification of bases in 16S rRNA essential for tRNA
binding at the 30S ribosomal P site. Science 267, 234–237 (1995).
24. Noller, H.F., Hoang, L. & Fredrick, K. The 30S ribosomal P site: a function of 16S
rRNA. FEBS Lett. 579, 855–858 (2005).
25. Herr, A.J., Atkins, J.F. & Gesteland, R.F. Mutations which alter the elbow region
of tRNA2Gly reduce T4 gene 60 translational bypassing efficiency. EMBO J. 18,
2886–2896 (1999).
26. Herr, A.J. et al. Analysis of the roles of tRNA structure, ribosomal protein L9, and the
bacteriophage T4 gene 60 bypassing signals during ribosome slippage on mRNA.
J. Mol. Biol. 309, 1029–1048 (2001).
27. Baranov, P.V., Gesteland, R.F. & Atkins, J.F. P-site tRNA is a crucial initiator of
ribosomal frameshifting. RNA 10, 221–230 (2004).
28. Schultz, D.W. & Yarus, M. tRNA structure and ribosomal function. I. tRNA nucleotide
27–43 mutations enhance first position wobble. J. Mol. Biol. 235, 1381–1394
(1994).
29. Hou, Y.M. & Schimmel, P. A simple structural feature is a major determinant of the
identity of a transfer RNA. Nature 333, 140–145 (1988).
30. McClain, W.H., Schneider, J., Bhattacharya, S. & Gabriel, K. The importance of tRNA
backbone-mediated interactions with synthetase for aminoacylation. Proc. Natl. Acad.
Sci. USA 95, 460–465 (1998).
31. Yarus, M. et al. The translational efficiency of tRNA is a property of the anticodon arm.
J. Biol. Chem. 261, 10496–10505 (1986).
32. Lustig, F. et al. The nucleotide in position 32 of the tRNA anticodon loop determines
ability of anticodon UCC to discriminate among glycine codons. Proc. Natl. Acad. Sci.
USA 90, 3343–3347 (1993).
33. Claesson, C. et al. Glycine codon discrimination and the nucleotide in position 32 of
the anticodon loop. J. Mol. Biol. 247, 191–196 (1995).
34. O’Connor, M. tRNA imbalance promotes -1 frameshifting via near-cognate decoding.
J. Mol. Biol. 279, 727–736 (1998).
35. Auffinger, P. & Westhof, E. An extended structural signature for the tRNA anticodon
loop. RNA 7, 334–341 (2001).
36. Dao, V. et al. Ribosome binding of DNA analogs of tRNA requires base modifications
and supports the ‘‘extended anticodon’’. Proc. Natl. Acad. Sci. USA 91, 2125–2129
(1994).
37. Cabello-Villegas, J., Winkler, M.E. & Nikonowicz, E.P. Solution conformations of
unmodified and A(37)N(6)-dimethylallyl modified anticodon stem-loops of Escherichia
coli tRNA(Phe). J. Mol. Biol. 319, 1015–1034 (2002).
38. Stuart, J.W. et al. Functional anticodon architecture of human tRNALys3 includes
disruption of intraloop hydrogen bonding by the naturally occurring amino acid
modification, t6A. Biochemistry 39, 13396–13404 (2000).
39. Shu, Z. & Bevilacqua, P.C. Isolation and characterization of thermodynamically stable
and unstable RNA hairpins from a triloop combinatorial library. Biochemistry 38,
15369–15379 (1999).
40. Battle, D.J. & Doudna, J.A. Specificity of RNA-RNA helix recognition. Proc. Natl. Acad.
Sci. USA 99, 11676–11681 (2002).
41. Pape, T., Wintermeyer, W. & Rodnina, M.V. Complete kinetic mechanism of elongation
factor Tu-dependent binding of aminoacyl-tRNA to the A site of the E. coli ribosome.
EMBO J. 17, 7490–7497 (1998).
42. Savelsbergh, A. et al. An elongation factor G-induced ribosome rearrangement
precedes tRNA-mRNA translocation. Mol. Cell 11, 1517–1523 (2003).
43. Atkins, J.F. et al. Poking a hole in the sanctity of the triplet code: inferences for
framing. in The Ribosome: Structure, Function, Antibiotics, and Cellular Interactions
(eds. Garret, R.A. et al.) 369–383 (American Society for Microbiology Press, Washington, DC, 2000).
44. Yarus, M. Translational efficiency of transfer RNA’s: uses of an extended anticodon.
Science 218, 646–652 (1982).
45. Yusupova, G.Z., Yusupov, M.M., Cate, J.H. & Noller, H.F. The path of messenger RNA
through the ribosome. Cell 106, 233–241 (2001).
46. Sampson, J.R. & Uhlenbeck, O.C. Biochemical and physical characterization of an
unmodified yeast phenylalanine transfer RNA transcribed in vitro. Proc. Natl. Acad.
Sci. USA 85, 1033–1037 (1988).
47. Wolfson, A.D. & Uhlenbeck, O.C. Modulation of tRNAAla identity by inorganic
pyrophosphatase. Proc. Natl. Acad. Sci. USA 99, 5965–5970 (2002).
NUMBER 9
SEPTEMBER 2005
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