Nucleic Acids Research, 1995, Vol. 23, No. 4
© 1995 Oxford University Press
683-688
Decoding with the A:l wobble pair is inefficient
James F. Curran
Department of Biology, Wake Forest University, Winston-Salem, NC 27109, USA
Received September 30, 1994; Revised and Accepted January 6, 1995
ABSTRACT
tRNAs with inosine (I) in the first position read three
codons ending in U, C and A. However, A-ending
codons read with I are rarely used. In Escherichia coli,
CGA/U/C are all read solely by t R N A ^ . CGU and CGC
ICG
are very common codons, but CGA is very rare. Three
independent in vivo assays show that translation of
CGA is relatively inefficient. In the first, nine tandem
CGA cause a strong r/?o-mediated polar effect on
expression of a lacZ reporter gene. The inhibition is
made more extreme by a mutation in ribosomal protein
S12 (rpsL), which indicates that ribosomal binding by
tRNA*rj> is slow and/or unstable in the CGA cluster.
ICG
The second assay, in which codons are substituted for
the regulatory UGA of the RF2frameshift, confirms that
aa-tRNA selection is slow and/or unstable at CGA. In
the third assay, CGA is found to be a poor 5' context for
amber suppression, which suggests that an A:l base
pair in the P site can interfere with translation of a
codon in the A site. Two possible errors, frameshifting
and premature termination by RF2, are not significant
causes for inefficiency at CGA. It is concluded that the
A:l pair destabilizes codon:anticodon complexes during two successive ribosomal cycles, and it is suggested that these properties contribute to the rare
usage of codons read with the A:l base pair.
INTRODUCTION
Transfer RNAs with inosine at the first (wobble) position are
widely distributed taxonomically, occurring in all bacteria and
eukarya for which representative sets of tRNAs have been
characterized (1). Inosine-containing tRNAs are predicted (2) to
translate three codons ending in C, U and A. The three base pairs
with inosine have different structures. C:I is Watson-Crick, and
U:I has the same geometry as a standard U:G pair. In contrast, A:I
is the only purine-purine pair used in decoding, and it must
therefore have a distinct structure. The two purines most likely
assume a 'long wobble' conformation, in which the N-glycosyl
bonds are separated by 1.5 A relative to other standard geometries
(2,3). Molecular modeling suggests that A:I may destabilize the
codon:anticodon complex (3).
Whether decoding with the A:I pair is efficient, or indeed
whether it occurs at all, has been widely debated. Crick's
predicted wobble rules (2) were based on determinations of which
tRNAs bound in vitro to ribosomes programmed with specific
trinucleotides. Clearly, CGA will allow binding by an inosinecontaining tRNA (1,4,5). But in those experiments binding
presumably occurred in the ribosomal P site, which does not
necessarily have the same specificity as the A site.
An experimental challenge to the prediction that inosine can
recognize adenosine was provided by Munz et al. (6), who show
that mutant strains of the yeast Schizosaccharomyces pombe are
not viable if the only tRNA available to decode UCA contains
first position inosine. There is also evidence that suggests that
Saccharomyces cerevisiae may also have difficulty translating
with A:I. In this yeast, the A-ending codons in family boxes have
two nominally cognate isoacceptors: one with inosine and one
with a modified uridine in the first position (1,7). This apparent
duplication of specificity may be necessary because inosine-containing tRNAs are not sufficiently active (7) at NNA codons.
Other evidence which suggests that A:I decoding may be
inefficient is that NNA codons putatively read with inosine are
rare in virtually all organisms (8). This apparently universal form
of codon bias may reflect general functional deficiencies for
decoding with the A:I base pair.
On the other hand, inosine-containing tRNAs must translate
A-ending codons in some organisms. Extensive characterizations
of the tRNA repertoires of the bacteria E.coli (9) and Mycoplasma
capricolum (10) and the yeast Candida cylindracea (11) show
that inosine-containing tRNAs are the only adapters available to
read certain NNA codons. Those examples clearly indicate that
the A:I pair can perform the essential decoding functions. It is not
known whether that decoding is efficient.
This work describes a study of in vivo decoding properties of
E.coli tRNA*r£, which is the only tRNA in E.coli that reads the
arginine codons CGU, CGC and CGA (CGH codons). It is shown
that translation of nine tandem CGA can cause a r/io-dependent
polar effect on gene expression. Furthermore, a mutation in the
ribosomal S12 gene (rpsL) strongly increases this inhibition of
expression. One reason for inefficient decoding is slow and/or
unstable binding to CGA in the A site. Another problem is that an
A:I pair in the P site interferes with translation of the next codon
in the A site.
MATERIALS AND METHODS
Strains and plasmids
All strains are E.coli K12. The primary strain was P90C (12),
which has the genotype del(lac-pro) ara thi. S90C (12) is an rpsLr
derivative of P90C. P90Crho is a rhol5 //v::TnlO derivative of
P90C. This strain was made by co-transduction of the rhol5
marker (UV sensitivity; 13) with the tetracycline resistance
marker specified by TnlO. The donor for the transduction was
SA2367, which was kindly provided by Sankar Adhya.
684
Nucleic Acids Research, 1995, Vol. 23, No. 4
Trans lot i ona1
Couple
ompA
Promoter
\
BamHI
I
lncZ
|Upsireom C l s t r o n |
pJC!105
o n colEl
B
bio
exhibit especially high codon usage bias (Table 1). Because
highly biased genes are typically highly expressed genes (23-25),
CGA may be especially rare in those genes because it does not
facilitate expression. Secondly, the other codons read solely by
tRNA£« are common (Table 1), and tRNA*r« is abundant (26).
Thus, use of the CGH codons is not limited by any general
deficiency of tRNA£r* such as low concentration. Instead, CGA
may be specifically avoided because translation of that triplet is
uniquely inefficient. Thirdly, the CGA triplet is not rare in the two
untranslated reading frames (Table 1), which shows that it does
not perturb gene or message structure. Instead, the rare occurrence of CGA only in the translated phase suggests that this triplet
has poor translational properties.
5 ' -GATCATTCGACGACGACGACGACGACGACGACGATG-3 '
Table 1. Occurrences of CGA and related codons
3 ' -TAAGCTGCTGCTGCTGCTGCTGCTGCTGCTGCCTAG-5 '
Figure 1. (A) Structure of the translationally-coupled lacZ vector. The upstream
cistron is translationally coupled to lacZ as described (13). (B) Sequence of the
overlapping 36mer oligonucleotides that encode the CGA9 repeat. The
CGU9-encoding oligos were identical, except for the necessary T+-M
exchanges.
Percentage
codons
In phase
Other phases
pJC1105 is diagramed in Figure 1. It was made by routine
methods; details will be provided on request. The region
containing the upstream cistron and translational couple to lacZ
are described (14). To make pCGA9 and pCGU9, oligonucleotides specifying the codon clusters were cloned into the BamHI
site in the upstream cistron. pGAC8 was derived from pCGA9 by
deleting one base 5' and two bases 3' of the CGA9 cluster. Those
changes cause the synthetic CGA repeat to be translated in the
'GAC' phase. Those changes were made by swapping in
restriction fragment cassettes taken from frameshift variants of
pJC 1105 (14). The lacZ/RF2 fusion constructs are described (15).
Plasmids encoding the /acZ-amber mutants were made by
cloning double-stranded oligos into the Hindlll and BamHI sites
of pJC27 (16). pM Y228 encodes Su7 under control of the lacuvS
promoter (17).
Assays
p-galactosidase assays were performed as described (18). Unless
otherwise stated the growth medium was Vogel-Bonner's
minimal salts (19) supplemented with 0.5% casamino acids
(Difco), 0.5% glucose, 100 (ig/ml proline and 20 ug/ml thiamine.
Relative lacZ message levels were measured by an SI nuclease
protection assay using a previously described method (20).
Probed was a 204 nucleotide fragment from near the 3' end of
lacZ (from nucleotides 1979 to 2183). As an internal hybridization control, 99 nucleotides at the 5' end 23S rRNA were also
probed.
RESULTS
CGA is a rare codon in E.coli
CGA is a rare codon, occurring at a frequency of only 0.35%
(from the ECO.COD file of the May, 1994 edition of the
TRANSTERM database; 21). Three observations suggest that
CGA is rarely used because of its translational properties. First,
CGA is used even less frequently (9-fold less) in genes that
CGA
0.04
CGU
3.5
CGC
1.8
dicodons
nCG Ann
1.9
nnC GAn
4.0
In-phase codon percentages are from the 1994 edition of the ECO_H.COD file
of the TRANSTERM database (20). For CGA in other phases, CGA was
counted within codon pairs in genes identified in the ECO_H.DAT file of the
1993 edition of the TRANSTERM database (21). That file identifies 106 unique
coding sequences that have high CAI values.
Clustered CGA codons inhibit expression
The common CGU and the rare CGA are both read solely by
tRNA^. To determine whether translation of CGA might be less
efficient than that of CGU, two constructs were made that specify
either nine CGA (pCGA9) or nine CGU (pCGU9). Constructs
were made by cloning double-stranded oligos into the same
BamHI site of pJCl 105 (Fig. 1; Materials and Methods). Except
for these nine codons, the constructs are identical. The synthetic
sequences are in a cistron placed upstream of a lacZ reporter. The
upstream cistron is translationally coupled to lacZ such that
P-galactosidase synthesis requires that ribosomes translate
through the upstream cistron, terminate, and then reinitiate at lacZ
(Fig. 1; 14). By having the upstream cistron translationally
coupled to lacZ, the p-galactosidase amino acid sequence does
not include the nine oligo-encoded arginines, which might have
interfered with assays for P-galactosidase activity. Control alleles
show that activity of the lacZ reporter is dependent on translation
of the upstream cistron (14).
In the standard E.coli host (P90C) pCGA9 gives 4.5-fold lower
p-galactosidase activity than pCGU9 (Table 2). The inhibitory
effect of nine CGA triplets is not due to its structure or low
stability because this sequence allows a high P-galactosidase
yield if translated in the overlapping 'GAC phase on pGAC8
(Table 2). That construct was made from pCGA9 by deleting one
base 5' and two bases 3' of the CGA repeat (Materials and
Methods). Dot blots show that pCGA9 and pGAC8 produce
similar levels of RNA specific for the CGA cluster (data not
shown), and thus support the conclusion that lacZ expression is
Nucleic Acids Research, 1995, Vol. 23, No. 4
23S
1
lac
I Plasmid and Host
PJC1105
P9OC
685
Ratio, lac/23S
0.43
pCGA9
"
0.16
pCGU9
"
0.4
PGAC8
"
0.49
pCGA9
P90Crho
0.28
pCGU9
"
0.48
Figure 2. Arrows point to the positions of the protected probes for 23S rRN A (23S) and lacZ mRNA (lac). The ratios of CPM in the lacZ band to the 23S band (Ratio,
lac/23S) were calculated from the scintillation counts from excised gel slices. The reported ratios are the averages of two assays, including those from the shown gel.
not limited by a low concentration of the message from the
upstream cistron. Below is described further evidence that
translation of the CGA cluster is inefficient.
Table 2. P-galactosidase activities of constructs with clustered codons
Construct
Host
P90C
S90C
P90Crho
pJC1105
1047 ±33
1196 ± 31
1350 ±32
pCGA9
107 ± 6
16± 1
431 ± 8
pCGU9
478+ 12
562 + 20
711 ±28
pGAC8
811 ±20
ND
ND
(5-galactosidase activities are the means of 6-10 assays ± standard errors of
means. ND means not determined.
Others have reported inhibitory effects by clusters of the
usually rare AGG (27-30). But in those cases, low expression
may be caused by the extremely low concentration of the cognate
tRNA (31,32). In the current experiment, because they are read
by the same tRNA, the difference between CGA and CGU cannot
be related to tRNA availability. Instead, the low activity of
pCGA9 may be related to the requirement for the A:I pair for
decoding.
The activities of pCGU9 and pGAC8 are lower than the vector
by factors of 2 and 1.5, respectively (Table 2). Mechanisms for
those effects were not explored. Instead, I concentrate on the
much larger difference between the pCGU9 and pCGA9
constructs.
expression of pCGA9 to near background but does not decrease
the activities of pCGU9 and the vector. Together with the
previous results, these data suggest that tRNAJ^ is unstable
and/or slow to act at the CGA cluster, and that rpsL exacerbates
this difficulty. This view is consistent with the observation (37)
that a synthetic sequence having many CGA interspersed within
it is translated relatively slowly in a wild type ribosomal
background. This apparent difficulty with CGA is most likely
associated with the requirement for translation with the A:I pair.
Clustered CGA invokes polarity
lacZ contains r/io-dependent transcriptional terminators (38), and
termination probability is inversely dependent on translational
efficiency (39-41). Thus it is conceivable that the inhibitory
effect of the CGA cluster is mediated by Wio-dependent
termination. To search for a polar effect, p-galactosidase activities
and lacZ message levels were compared between rho+ and rho~~
strains. Probed was a 204 nucleotide section of lacZ message
from the a region about 1500 nucleotides downstream of known
r/io-dependent terminators (38).
In the rho+ host (P90C) pCGA9 does indeed produce a low
level of lacZ message, relative to other constructs (Fig. 2). The
rho-15 mutation substantially increases lacZ message from
pCGA9 (Fig. 2). In addition, the rho-15 mutation also increases
the P-galactosidase activity from pCGA9 so that it is only 1.5-fold
lower than that from pCGU9 (Table 2). Together these data
indicate that the CGA cluster inhibits lacZ expression by a polar
mechanism.
pCGA9 is sensitive to an rpsL mutation
CGA competes relatively poorly with the RF2
frameshift
As another test for a translational phenotype for the CG A9 cluster,
the effect of a streptomycin resistance mutation in rpsL was
examined. Streptomycin resistance mutations in rpsL increase the
stringency of aa-tRNA selection (33,34). For example, such
mutations generally depress nonsense suppression efficiency
(33). In vitro studies suggest that initial selection of EF-Tu»aatRNA«GTP complexes is inhibited by rpsL mutations (35,36).
Proofreading may also be increased by rpsL mutations (34,35).
S90C was transformed with either pCGA9, pCGU9 or the
vector (pJC1105), and the p-galactosidase activities of isolates
were measured (Table 2). The rpsL mutation reduces the
The CGH codons were substituted for the regulatory UGA at the
E.coli RF2 frameshift site in lacZMFl fusions such that
p-galactosidase activity requires frameshifting (15). At this site,
frameshifting and in-phase translation of the codon are competing
reactions, and frameshift-dependent P-galactosidase activity is
predicted to be inversely related to the rate of aa-tRNA selection
(16). We and others have confirmed that frameshifting competes
with normal translation by showing that increased aa-tRNA
activity decreases frameshifting (15,16,42,43). Frameshift frequency, therefore, can be used to estimate relative rates of
aa-tRNA selection (15,16,20).
686
Nucleic Acids Research, 1995, Vol. 23, No. 4
In this assay, CGA allows more frameshift-dependent p-galactosidase activity than either CGU or CGC (Table 3), which suggests
that CGA is slower to select tRNA£« than are its synonyms.
When P-galactosidase activities are normalized for lacZ message
levels, the codons differ in rate of aa-tRNA selection in the order
CGU > CGC > CGA. This order correlates with codon frequency
in highly biased genes (Table 1).
Table 3. Frameshift-dependent P-galactosidase activities
Construct
Host P90C
p-gal
pJC27
P-gal/mRNA
P-gal
1.8
6936
10 2901 140
pRF/CGA
689 ± 19
0.43
1602
1427 ±46
pRF/CGC
356 ± 8
0.34
1047
409 1 42
345 ± 10
0.39
885
434 ± 12
pRF/CGU
12484±405
Host S90C
mRNA
'P-gal' is P-galactosidase activity, and is reported as in Table 1. 'mRNA' is the
ratio of /acZ-specific CPM divided by 23S rRNA-specific CPM as in the legend
to Figure 2. Reported are the averages of four determinations; all standard errors
the means are <10%. 'P-gal/mRNA' is the ratio of the two determinations.
This order differs from that reported earlier (15). In the earlier
work, the codons differed by about the same factors but in the
order CGU > CGA > CGC. I attempted to track the source of the
discrepancy. The current assay differs from the old one in two
ways: I now use a rec+ host and a richer growth medium
(Materials and Methods). I reassayed the CGH alleles using the
old system and observe p-galactosidase values virtually identical
to those in Table 3. Therefore, I conclude that the current values
are representative. The simplest explanation for the discrepancy
is that in the earlier work the cultures for CGA and CGC, which
were assayed during the same period, were inadvertently
switched. To determine whether other data in the earlier work
might not be representative, I redetermined P-galactosidase
activities of all of the other strains used previously. All of the other
strains give essentially the same P-galactosidase activities as
before (data not shown).
Sipley and Goldman (42) observed that rpsL can reduce rates
of aa-tRNA selection as measured with this assay. The rpsL
mutation increases frameshift-dependent P-galactosidase activities at all three codons (Table 3), but the difference between
CGA and the others increases from 2- to 4-fold. Together, the
results in this section suggest that the requirement for an A:I base
pair slows and/or destabilizes tRNA£« at CGA. It seems likely
that this defect contributes to the polar effect caused by the
pCGA9 cluster.
CGA interferes with translation of the next codon
The third position base pair in the P site may affect the A site
message:anticodon complex (44-46). It seems plausible, then,
that the unusual A:I pair will exert a context effect on the reading
of the next codon. To provide a fully controlled assay for A:I 5'
context effects, three lacZ alleles were constructed with each
CGH codon 5' to an amber codon. During suppression of the
amber codon in the A site, the peptidyl- tRNA^| anticodon is
base paired to the corresponding CGH triplet. Except for the base
pair with inosine, all components of the ribosomal complexes,
including all mRNA and tRNA nucleotides, and even the nascent
polypeptide, are absolutely identical. Therefore, any differences
in suppression efficiency among these alleles must result from
structural/functional differences of the base pairs with inosine.
Suppression is greatest at the C:I context, with U:I slightly
below (Table 4). In contrast, the allele with the A:I context has a
2.5-3-fold lower suppression efficiency. Others observe that
CGA is a poor context for UGA suppression (Leif Isaksson,
personal communication). These data strongly suggest that the P
site wobble position affects reading by the suppressor tRNA, with
A:I being inhibitory. An alternative, that the A:I pair enhances
termination by RF1, is unlikely. Context is strongly biased at
natural termination sites (21,47), and preferred contexts enhance
RF1 action (20). But CGA is an extremely rare 5' context at
natural UAG (21), which suggests that A:I does not stimulate
RF1. Instead, the low efficiency of the CGA allele probably
results from interference with the suppressor by the P site A:I pair.
It is likely that this problem contributes to the polar effect of
pCGA9.
Table 4. CGH context effects on amber suppression
Plasmid
p-galactosidase activity
pJC27
10 7061460
pCGA UAG
10741 110
pCGC UAG
2992 1 320
pCGU UAG
2585 ± 90
p-galactosidase activities are as on Table 1. Cells contain the Su7 amber suppressor expressed from pMY228 (16).
CGA is not prone to frameshift or to premature
termination by RF2
Two translational reactions, frameshifting and premature termination by RF2 (i.e., by misreading CGA as UGA), do not
contribute to the low activity of CGA9. Frameshifting is ruled out
by the very low activities of frameshift constructs in which one
or two bases are deleted 3' of the CGA9 cluster. These constructs
give 38 and 22 P-galactosidase units, respectively. Those low
activities argue that ribosomes do not frequently frameshift
during translation of the CGA cluster.
Action by RF2 is ruled out because lacZ expression from
pCGA9 is unaffected by overexpression of RF2 (data not shown;
RF2-encoding plasmids were kindly provided by Warren Tate,
University of Otago, New Zealand).
DISCUSSION
The A:I base pair makes translation inefficient. One problem is
slow and/or unstable aa-tRNA binding at the ribosomal A site.
Another problem is that an A:I pair in the P site interferes with
translation of the next codon. Thus, the A:I pair makes translation
inefficient for at least two ribosomal cycles. It is very likely that
these poor translational phenotypes are at least partly responsible
for the very rare occurrence of CGA codons in E.coli (Table 1).
These phenotypes may also contribute to the rare use of codons
read with the A:I pair in virtually all organisms (8).
Codon usage that limits the translational problems of A:I
decoding may even be vital. Wild type Schizosaccharomyces
pombe has two tRN As specific for the UCA codon (1), and this
yeast is not viable if mutations leave it with only the inosine-
Nucleic Acids Research, 1995, Vol. 23, No. 4
containing isoacceptor (6). Though it remains formally possible
that the missing tRNA is needed for some vital non-translational
function, the simplest interpretation is that the inefficient A:I
interaction is unable to fully support translation of UCA in this
yeast. But UCA is not rare in S.pombe (21); at a frequency of
1.6%, UCA appears several times in the typical gene. It is possible
that the synthesis of essential proteins is inhibited by a large
number or unfortunate locations of sites that must be translated
with the inefficient A:I pair.
Genetic, physical and theoretical studies suggest that the third
base pair of the P site codon-anticodon complex may interact
with the A site (for reviews, see 45 and 46). It may not be
surprising, therefore, that the large A:I pair is a poor 5' context for
amber suppression (Table 4). Exactly how a P site A:I pair
interacts with the A site will depend on the structures of the active
coding sites. The available data cannot distinguish between two
coding site configurations that differ in the relative orientation of
the P and A sites (see 45 and 46). I have evaluated published
figures of both configurations to determine how each might
respond to an A:I wobble pair in the P site. In the'S' configuration
(45,46), the P site anticodon loop expands towards the A site so
that it might directly affect decoding. In the 'R' configuration
(3,45), the P site anticodon loop expands in parallel with the A site
so that direct effects on decoding are less likely in this
configuration. Of course, indirect effects through the ribosome
are possible in the 'R' model. Thus, proof of the relative
orientation of the coding sites will also determine whether a P site
A:I pair interacts directly or indirectly with the A site.
Many other studies also show that third base pair structure is a
context determinant. For example, Kato et al. (48) show that the
U:A Watson-Crick base pair is a better 5' context than U:G for
the transpeptidation reaction on poly-U programmed ribosomes.
The current data also hints that C:I, which is Watson-Crick, is
better than U:I, which resembles U:G; however, the difference
between these two contexts is barely significant (Table 4). Stormo
et al. (49), from correlations between suppression efficiency and
flanking message nucleotides, show that A and C are better 5'
contexts for amber suppression. In that study, the 5' flanking
position is studied as a nucleotide rather than a base paired
structure, but A and C are usually decoded by Watson-Crick
pairing. Thus these data also suggest that third base pair structure
affects the next codon. (A:I is a notable non-Watson-Crick pair
to message A; there were no A:I contexts in that study.) Finally,
Folley and Yarus (50) show that repeated use of the same third
position base pair (either A:V or G:V, where V is 5-oxyacetic
uridine) for several codons can reduce expression of a lacZ
reporter.
The current data together with those other studies suggest a
molecular rationale for certain patterns of message context bias
(for reviews, see 51 and 52). Because these context effects are
related to the structure of the third position base pair, they may be
an inherent consequence of wobble decoding. Third position
effects may, therefore, contribute to the strong, pervasive biases
in dinucleotide frequencies at codon interfaces (3-1 bias; 53-57)
or between adjacent wobble positions (3-3 bias; 53-55).
Inhibition of the CGA9 cluster is a r/io-dependent polar effect.
The most probable mechanism is that slow translation of the
CGA9 cluster uncouples translation from transcription, and that
the resulting untranslated transcript stimulates r/io-mediated
termination (38,58). Folley and Yarus (50) previously concluded
that certain codon contexts slow ribosomal progression and
687
thereby cause polarity, but polarity was not directly shown. The
current data clearly indicate that codon and/or context usage
affect r/io-mediated termination. Latent intragenic terminators
are common (59,60), and one role may be to prevent message
synthesis during amino acid limitation (60). That codons and
contexts affect termination suggests that terminators may be
coordinated with codon usage. This may help explain the strong
tendency for rarely used codons to occur very early in genes
(29,61). Rare codons may increase terminator sensitivity to
nutritional downshifts, as they do at canonical attenuators (62,
63).
ACKNOWLEDGEMENTS
I am grateful to Glenn Bjork and Amanda Karper for helpful
comments and to Leif Isaksson for unpublished data. I am
especially grateful to Sankar Adhya, Liz Poole and Warren Tate
for materials, and to Chris Brown for help with the TRANSTERM database. The work was facilitated by the DNA Synthesis
Laboratory of the Comprehensive Cancer Center of Wake Forest
University. This work was supported by NSF grant
DMB-8904708 and by a Z. Smith Reynolds Foundation faculty
development award administered by Wake Forest University's
RECREAC Program.
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