Control of translation efficiency in yeast
by codon–anticodon interactions
DANIEL P. LETZRING, KIMBERLY M. DEAN, and ELIZABETH J. GRAYHACK
Department of Biochemistry and Biophysics, University of Rochester Medical School, Rochester, New York 14642, USA
The choice of synonymous codons used to encode a polypeptide contributes to substantial differences in translation efficiency
between genes. However, both the magnitude and the mechanisms of codon-mediated effects are unknown, as neither the
effects of individual codons nor the parameters that modulate codon-mediated regulation are understood, particularly in
eukaryotes. To explore this problem in Saccharomyces cerevisiae, we performed the first systematic analysis of codon effects on
expression. We find that the arginine codon CGA is strongly inhibitory, resulting in progressively and sharply reduced
expression with increased CGA codon dosage. CGA-mediated inhibition of expression is primarily due to wobble decoding of
CGA, since it is nearly completely suppressed by coexpression of an exact match anticodon-mutated tRNAArg(UCG), and is
associated with generation of a smaller RNA fragment, likely due to endonucleolytic cleavage at a stalled ribosome. Moreover,
CGA codon pairs are more effective inhibitors of expression than individual CGA codons. These results directly implicate decoding
by the ribosome and interactions at neighboring sites within the ribosome as mediators of codon-specific translation efficiency.
Keywords: yeast; translation; genetic code; ribosome; codons; tRNA
Differences in translation efficiency contribute substantially
to the large variation in protein expression, with translation
of individual mRNAs ranging over at least three orders of
magnitude in the yeast Saccharomyces cerevisiae (Arava et al.
2003; de Godoy et al. 2008; Ingolia et al. 2009). While
differential translation efficiency is the result of many fac-
tors, substantial evidence from both bacteria and eukary-
otes indicates that the amount of polypeptide produced per
mRNA is influenced by the particular choice of synonymous
codons, codons that specify insertion of the same amino acid
(Ghaemmaghami et al. 2003; Brockmann et al. 2007).
In organisms such as Escherichia coli, S. cerevisiae,
Caenorhabditis elegans, and Drosophila melanogaster, a sub-
set of synonymous codons, generally those decoded by the
most abundant tRNAs without wobble decoding (Ikemura
1982), are ‘‘preferred codons’’ for efficient translation and
are used more frequently than their synonyms (dos Reis
et al. 2004; Parmley and Hurst 2007). Preferred codons are
used nearly exclusively in many highly expressed genes,
sharply differentiating these genes from the majority of
other genes (Grantham et al. 1980; Bennetzen and Hall
1982; Duret and Mouchiroud 1999). Indeed, codon usage
and protein abundance correlate genome-wide in S. cer-
evisiae and E. coli (Ghaemmaghami et al. 2003; Tuller et al.
2007; Ishihama et al. 2008; Tuller et al. 2010b). Remarkably,
despite changes in the actual identity of the preferred
codons, the selection for preferred codons in many gene
families is maintained across species (Man and Pilpel 2007).
In addition, expression of genes in E. coli and in S. cerevisiae
is frequently improved by recoding genes with preferred
codons (Gustafsson et al. 2004; Burgess-Brown et al. 2008;
Keppler-Ross et al. 2008; Quartley et al. 2009; Welch et al.
2009), or by overproducing rare tRNAs in E. coli (Burgess-
Brown et al. 2008).
Nonetheless, the degree to which codons affect expres-
sion in E. coli has been the topic of some debate, with two
recent papers arriving at very different conclusions about
the quantitative importance of codon effects. Kudla et al.
(2009) concluded that codon bias had little significant
effect on protein levels of GFP variants, while Welch et al.
(2009) found that variation in expression of two genes was
strongly correlated with codon biases for 10 amino acids.
However, it is likely that the coupling between transcription
and translation complicates the analysis of codon effects in
bacteria, since some codon changes are known to cause
premature rho-mediated transcription termination (Deana
Reprint requests to: Elizabeth J. Grayhack, Department of Biochemistry
and Biophysics, University of Rochester School of Medicine and Den-
tistry, 601 Elmwood Ave., Rochester, NY 14642, USA; e-mail: elizabeth_
email@example.com; fax: (585) 272-2766.
Article published online ahead of print. Article and publication date are
RNA (2010), 16:2516–2528. Published by Cold Spring Harbor Laboratory Press. Copyright ? 2010 RNA Society.
et al. 1998). This difference in the molecular basis for codon
effects between bacteria and eukaryotes is an argument to
examine the role of codon usage in a eukaryote.
Assessing the contribution of codons to gene expression
in eukaryotes still has at least two difficulties. First, syn-
onymous codon choice influences several aspects of trans-
lation, including the accuracy of amino acid insertion
(Kramer and Farabaugh 2007), reading frame maintenance
(Farabaugh et al. 2006), and folding of the nascent poly-
peptide (Kimchi-Sarfaty et al. 2007), each of which may
differentially affect the stability or activity of the resulting
protein. Second, synonymous codon choice affects the
mRNA sequence, thereby affecting mRNA structure, which
itself directly affects translation (Kudla et al. 2009). The
mRNA sequence also influences the binding of proteins
and RNAs that regulate splicing, decay, and translation effi-
ciency (Doma and Parker 2006; Isken and Maquat 2007).
Although the preferred codons are well characterized,
neither the identity nor the properties of codons or codon
combinations that cause reduced expression in eukaryotes
are known. Codons that impair expression in yeast are
difficult to decipher because the 37 unpreferred codons
have different properties, with some decoded by rare
tRNAs, others requiring wobble decoding, and still others
are simply underused. Moreover, it remains controversial
whether the mediator of reduced expression is individual
codons, adjacent codons (codon pairs), or clusters of rare
codons (Smith and Yarus 1989; Irwin et al. 1995; Kane
1995; Moura et al. 2005; Buchan et al. 2006; Coleman et al.
2008). Knowledge of the identity and properties of codon-
initiated inhibitory signals is required to understand the
underlying mechanisms for this regulation.
Here we describe the first systematic study of codon
effects on expression. We assessed the effect of each of 59
codons in the yeast S. cerevisiae by monitoring firefly lucif-
erase activity of constructs with repeats of individual codons.
We identify the Arg codon CGA as strongly inhibitory, an
effect due almost entirely to IdA wobble decoding of the
CGA codon. We find that CGA codons give rise to a stable
mRNA fragment containing sequences downstream from
the CGA codons, from which we infer that these codons
likely cause arrest of the ribosome. Moreover, we find that
adjacent CGA codons are much more potent inhibitors of
expression than separated CGA codons. Thus, we conclude
that translation efficiency is directly modulated by decod-
ing interactions within the ribosome.
In a systematic analysis, most codons exert only slight
effects on expression
To assay the effects of sequences containing codon repeats,
we inserted them into common flanking sequences, either
near the N-terminus at amino acid 4 of the firefly luciferase
gene (Grentzmann et al. 1998; Keeling et al. 2004) (referred
to as X4F), or at amino acid 314 between the fused Renilla
and firefly luciferase genes (RX314F) (Fig. 1A). We examined
FIGURE 1. Systematic screen of the effects of synonymous codon repeats on firefly luciferase expression. (A) Schematic of reporter constructs
used in these studies: Codon repeats (X) were inserted upstream of a firefly luciferase reporter gene either at position 4 of the coding region (X4F),
at position 314 in the Renilla-firefly fusion construct (RX314F) or at position 4 of the coding region of Renilla luciferase (X4R) or superfolder GFP
(X4G) (Pedelacq et al. 2006). X4F and RX314F were used for a systematic screen of the effects of 59 codons on expression. (B) Inhibitory effects
of several codon repeats, but not CGA repeats, in X4F are likely due to formation of strong RNA secondary structures. Luciferase activity for each
10-mer codon repeat at the N-terminus (%C) is plotted as a function of the free energy of the predicted RNA secondary structure from 1 to 50.
Free energy of folding (nucleotides 1–50 of X4F-reporter constructs containing 10 repeats of a particular codon for 59 of the 61 sense codons)
(kcal/mol) was calculated using RNAstructure (Mathews et al. 1999) version 4.6 (http://rna.urmc.rochester.edu/RNAstructure.html). Similar
results were obtained from calculations of free energy of folding from ?4 to +37.
Decoding interactions impair translation
the effect of repeats of 10 identical
codons in both positions, for 59 codons
(except [CCC]10and [GGG]10for tech-
nical reasons). We also examined the
effect of repeats of four identical codons
in the N-terminal position, for 23 co-
dons, specifying nine amino acids that
were found to be inhibitory with all
specifying codons in the 10-mers. Rela-
tive luciferase activity of yeast expressing
each of these constructs was assayed and
reported, both with respect to expression
from a vector with no insert (%V), and
with respect to the identical polypeptide
encoded by the synonymous codon that
gave maximal activity (%C) (Table 1).
We draw three conclusions from the
analysis of the 57 codons with one or
more synonymous codons. First, most
synonymous codons have little effect on
expression in these reporters. Most con-
structs yielded >66%C activity, includ-
ing 36 and 51 of 57 codon repeats with
10 amino acids in X4F and in RX314F,
respectively, and 20 of 23 codon repeats
with four amino acids in X4F. As ex-
pected, most of the codons identified as
preferred codons in S. cerevisiae (Jansen
et al. 2003) yielded high expression (19
of 22 in X4F and 22 of 22 in RX314F)
(see Table 1).
Second, the inhibitory effects of many
codon repeats in X4F are likely due to
formation of secondary structures in the
mRNA near the AUG, and thus may not
reflect codon effects. For 15 codon re-
peats, we find that poor expression of
X4F correlates with predicted thermody-
namic stability in the first 50 bases (fold-
ing free energy less than ?16 kcal/mol)
(Fig. 1B). Since strong secondary struc-
tures near the 59 end are inhibitory in
E. coli (Kudla et al. 2009) and are selected
against in both E.coli and S. cerevisiae
(Tuller et al. 2010b), we infer that sec-
ondary structure may explain some or all
of the inhibitory effects of these codon
repeats in the N-terminal location, and,
thus, these codons were not further an-
alyzed. Similarly, the effects of Lys AAA
(24%C) are likely due to the polyA se-
quence, which is known to inhibit ex-
pression (Dimitrova et al. 2009).
Third, Arg CGA was strongly inhibi-
tory both in the N-terminal and internal
TABLE 1. Systematic analysis of codon effects on firefly luciferase activity
Codon Amino acid % V%C % V%C%V%C
Letzring et al.
RNA, Vol. 16, No. 12
positions (Fig. 2A; Table 1). In the N-terminal position, four
other sequences likely encode inhibitory codons (Fig. 1B;
Table 1,), including Thr ACG, Val GUA, and possibly Ser
UCG codons, which are decoded by essential single (or two
copy) tRNAs (Johansson et al. 2008) and Ser AGU, which,
like Arg CGA, is decoded by wobble decoding. In the
internal position, four other Arg codons, as well as Phe
UUU, yielded <66%C (Fig. 2A; Table 1).
CGA codon repeats severely inhibit expression
in a dose-dependent manner
The Arg CGA codon stood out as a candidate for a strongly
inhibitory codon, because of both the magnitude and gen-
erality of its effects on expression. The CGA codon repeats
were strong inhibitors of expression in both positions, with
10 codon repeats resulting in 0.5%C and 1%C firefly
luciferase activity, respectively (Fig. 2A). CGA repeats also
exerted strong inhibitory effects upstream of Renilla lucif-
erase (Fig. 2A). Although all of the unpreferred Arg codon
repeats were inhibitory, none affected all three reporters to
nearly the same extent as CGA.
The effects of CGA codons on expression are unlikely to
be due to RNA secondary structure for two reasons. First,
the predicted free energy of the CGA repeat (?10.2 kcal/
mol) is inconsistent with an inhibitory RNA secondary
structure (Fig. 1B). Second, CGA repeats in the other read-
ing frames did not cause similar effects. The Asp GAC and
Thr ACG codons, which are permutations of CGA, form
repeats of the same sequence, and thus, likely form struc-
tures similar to (CGA)10, but neither codon repeat inhibits
expression nearly as much as CGA, either in terms of abso-
lute, or polypeptide-adjusted, levels of luciferase (Table 1).
We also found that very few CGA codons are required
for severe inhibition of expression. First, to determine the
extent to which CGA codons affect expression, we exam-
ined the expression of firefly luciferase as a function of
CGA codon dosage. In each case, we made identical Arg10-
containing luciferase proteins, with the
Arg residues coded by increasing num-
bers of CGA codons (Fig. 2B). In this con-
text, two CGA codons in the N-terminal
position caused a decrease in firefly
luciferase activity to 46%C, while four
CGA codons caused a decrease to 12%C
(Fig. 2C). Within the first 14 amino
acids, the position of the codons did
not matter, as the constructs (CGA)5–
(AGA)5 and (AGA)5–(CGA)5 yielded
similar levels of luciferase activity (see
Fig. 7B below). Furthermore, CGA co-
dons were also strongly inhibitory in
the internal location (RX314F), although
slightly more CGA codons were re-
quired to exert similar levels of inhibi-
tion in this position compared to the N-terminal location
(Fig. 2C). Second, the inhibitory effects of CGA were
apparent with only three or four Arg residues. Inhibition
by CGA codons (5.2%C) is observed with only four Arg
residues upstream of firefly luciferase (Fig. 2D; Table 1),
and three arginines upstream of GFP (6.7%C) (Fig. 2E).
Thus, inhibition by CGA is effective with very few codons
and does not require an extended arginine repeat.
Inhibition by CGA is due to IdA wobble decoding
To determine explicitly if the CGA codon inhibits expres-
sion, we examined the effect of overproducing the corre-
sponding tRNA species on expression of (CGA)10constructs.
CGA codons are decoded by tRNAArg(ICG), which is encoded
by six tRNAArggenes (Fig. 3A) bearing an ACG anticodon
that is subsequently converted to ICG (Grosjean et al. 2010).
Because the number of gene copies of tRNAs vary from one
to 17, and the amounts of various tRNA species correspond
closely to the number of genes that specify each variant
(Tuller et al. 2010a), these six copies should yield sufficient
amounts of tRNA to decode CGA. Nonetheless, overpro-
duction of tRNAArg(ICG), but not any other Arg isoacceptor,
resulted in an z10-fold increase in X4F–(CGA)10luciferase
activity compared to the vector control (Fig. 3B), and sim-
ilarly suppressed the inhibitory effect of the internal CGA
codon repeat (Fig. 3C). This increase in expression to z7%
of X4F–(AGA)10luciferase activity (Fig. 3B) was as much as
would be expected from the likely increase in the con-
centration of tRNAArg(ICG)due to the tRNA gene on the
multicopy plasmid, since the multicopy plasmid added z25
copies of the tRNAArg(ICG)gene (Schneider and Guarente
1991) to the six genomic copies, resulting in an approxi-
mately fivefold increase in gene copy number. Thus, we
conclude that some of the CGA-mediated reduced ex-
pression is due to a defect in translation of CGA codons.
Since suppression by the overproduced tRNAArg(ICG)was
incomplete, we inferred that either a portion of the CGA
TABLE 1. Continued
X4F-10 mer RX314F-10merX4F-4mer
CodonAmino acid % V%C % V %C %V%C
Firefly luciferase activity of X4F and RX314F constructs with insertion of either 10 or four
copies of each codon, as indicated. Two values are reported for each construct: (%V) is the
percent activity with respect to the wild-type vector without an insertion, and (%C) is the
percent activity with respect to the maximum activity obtained with each amino acid
insertion. Optimal codons, as reported by Jansen et al. (2003), are shaded and bolded. (nd =
Decoding interactions impair translation
effects is not codon-dependent, or that
decoding of the CGA codon by its
cognate tRNAArg(ICG)is inherently ineffi-
cient. Decoding of the CGA codon might
be inefficient either due to inherently
poor tRNA function of tRNAArg(ICG)or
due to the IdA wobble decoding neces-
sary to decode the CGA codon. To ad-
dress these issues, we constructed two
tRNAs that should exactly base pair (an
anticodon-match) to the CGA codon,
by mutating the anticodon of the major
tRNAArg(UCU)species from UCU to UCG,
and of tRNAArg(ICG)from ACG to UCG.
The introduction of an anticodon-
matched isoaccepting tRNA almost
completely suppressed the expression
defect caused by the 10 CGA codons.
Overproduction of either the mutant
tRNAArg(UCU to UCG)or the mutant
tRNAArg(ICG to UCG)resulted in a 60-
fold increase in luciferase activity, to
nearly the levels of X4F–(AGA)10(Fig.
4A). Similar suppression was observed
for the reporter with CGA codons at
amino acid 314 (Fig. 4B). This result
demonstrates that almost all effects of
the (CGA)10sequence are due to the use
of this codon in translation, suggests
that CGA decoding is severely deficient,
and implies that IdA wobble decoding
is a prominent factor in CGA effects on
To evaluate the importance of tRNA
abundance in decoding CGA with an
exact match tRNA, we examined sup-
pression of CGA-mediated inhibition of
expression by a single copy of the anti-
codon mutated tRNA genes integrated
into the yeast chromosome (Fig. 4C,D).
Remarkably, we found that an integrated
copy of either tRNAArg(ICG to UCG)or
tRNAArg(UCU to UCG)strongly suppressed
the expression defect caused by CGA
codons, resulting in a 40- to 50-fold im-
provement in expression of the (CGA)10
construct (Fig. 4D). Similarly, expression
of a (CGA)5(AGA)5luciferase construct
improved z20-fold in strains expressing
these exact match single copy tRNAs
(Fig. 4C). The simplest interpretation
of this strong suppression is that the
exact match tRNAArg(UCG)decodes a dis-
proportionate fraction of CGA codons
relative to that decoded by the more
FIGURE 2. CGA codon repeats inhibit expression in a dose-dependent manner. (A)
Luciferase activity of three different reporter constructs (X4F, RX314F, and X4R), containing
(Arg)10insertions specified by each of the six Arg codons. (B) The relationship between CGA
codon dose and luciferase activity was examined with (Arg)10inserts containing increasing
CGA content, as illustrated in the schematic. (C) Firefly luciferase activity as a function of CGA
codon dose in X4F (D) and RX314F (j)—reporter constructs. (D) Firefly luciferase activity of
X4F-reporter constructs containing either no insertion, or insertion of four Arg residues,
specified with the indicated CGA or AGA codons. Values were normalized to the X4F-(AGA)4
reporter. (E) Expression of integrated GFP constructs with three Arg residues inserted at
amino acid 4 specified by AGA or CGA codons. GFP expression was assessed based on the
distribution of yeast cells exhibiting different intensities of fluorescence emission at 530 nm.
Letzring et al.
RNA, Vol. 16, No. 12
abundant tRNAArg(ICG). Thus, the decoding properties of the
exact match tRNAArg(UCG), not its abundance, are responsi-
ble for efficient expression.
CGA codons do not result in reduced mRNA amounts,
but do yield a shortened RNA
Defects in translation are often closely coupled to mRNA
degradation (Frischmeyer et al. 2002; van Hoof et al. 2002;
Inada and Aiba 2005; Doma and Parker 2006; Isken and
Maquat 2007) and sometimes also affect protein folding
and activity (Kimchi-Sarfaty et al. 2007). To directly assess
mRNA and protein levels, we introduced an RGS-(His)6
epitope tag upstream of an (Arg)8 insertion, coded by
(AGA)8or increasing increments of CGA up to (CGA)8.
The introduction of progressively more CGA codons
resulted in reduced firefly luciferase activity (Fig. 5) and
in a parallel loss of protein (Fig. 5). Firefly luciferase
protein was weakly detectable from strains bearing the
(CGA)3(AGA)5and (CGA)4(AGA)4constructs, which pro-
duced 8%C–12%C luciferase activity, and was undetectable
from strains bearing the (CGA)8constructs. However, we
did not observe concurrent loss of firefly luciferase mRNA
from any of the (CGA)-containing constructs, relative to
that from constructs with (AGA)8, using an RT-PCR assay
with the indicated probes (Fig. 5).
To ascertain whether mRNA stability of transcripts
bearing CGA codons is a general phenomenon and to
examine the RNA species more carefully, we also examined
Renilla and firefly luciferase mRNAs by Northern analysis
of RNA from strains bearing the RGS-(His)6-(Arg)8in-
sertions at either amino acid 4 of Renilla luciferase (X4R) or
at amino acid 314 in the Renilla-firefly luciferase fusion
(RX314F). In both cases, we observed that the amount of
full-length luciferase RNA from the (CGA)8construct is
similar to (or slightly greater than) that from the corre-
sponding (AGA)8 construct (Fig. 6B,F). Neither the
amount nor the approximate size of Renilla luciferase
mRNA from the X4R reporter was reduced in RNA from
strains bearing either the (CGA)4(AGA)4 or the (CGA)8
constructs, relative to that of the (AGA)8 or no-insert
constructs (Fig. 6A,B). In fact, the CGA-containing mRNAs
were approximately twofold more abundant (Fig. 6B), a phe-
nomenon that was not observed if tRNAArg(ICG to UCG)was
coexpressed (Fig. 6C). As expected, the amounts of Renilla
luciferase protein were reduced by CGA codons, and restored
by coexpression of the exact match tRNAArg(UCG)(data not
shown). Likewise, the large RNA from the RX314F fusion that
hybridized to both the Renilla luciferase and firefly luciferase
probes was approximately twofold more abundant in strains
bearing constructs with (CGA)8, compared to strains bearing
constructs with (AGA)8, [(CGA)4(AGA)4], or no insert (Fig.
6F). Furthermore, we note that Renilla luciferase mRNA is
itself suitable for RNA degradation, since introduction of
either a UAA or a UGA stop codon at amino acid 4, which
would be expected to elicit nonsense-mediated decay in sus-
ceptible genes (Isken and Maquat 2007), results in reduction
of Renilla luciferase mRNA to z32% of the no-insert con-
trol (Fig. 6D). Thus, we conclude that CGA-mediated inhi-
bition of expression is not generally accompanied by mRNA
In addition to the full-length RLuc-FLuc fusion RNA,
we also detected a smaller RNA species from strains ex-
pressing either the (CGA)8or the [(CGA)4(AGA)4] RX314F
reporters (Fig. 6E,F). If the ribosome stops or stalls at CGA
codons, the mRNA might undergo cleavage, since many
stalls during translation trigger endonucleolytic cleavage
near the site at which the ribosome stalls (Doma and Parker
2006). The smaller RNA (labeled FLuc mRNA 2 in Fig. 6F),
had the properties expected for an RNA cleaved at or near
the CGA codons. This smaller RNA was detected with the
firefly luciferase probe, but not the Renilla luciferase probe,
and was found only in strains bearing the (CGA)8or
[(CGA)4(AGA)4] constructs (Fig. 6F). Moreover, there is
significantly more small RNA from strains with the (CGA)8
reporters than in the [(CGA)4(AGA)4] reporters. We did
FIGURE 3. Suppression of CGA-mediated expression defects by its cognate tRNA. (A) The Arg codons are compared with respect to use in the
genome, gene copy number of the decoding tRNAArgspecies and the requirement for wobble decoding. (B) Effect of overproduction of the
indicated tRNAArgspecies from high copy plasmids on firefly luciferase activity of X4F-reporter constructs containing (CGA)10insertions. Values
were compared to activity of X4F-(AGA)10luciferase in a strain with an empty vector. (C) Effect of overproduction of tRNAArg(ICG)from a high
copy plasmid on firefly luciferase activity of RX314F-reporter constructs containing (CGA)10insertions at amino acid 314. Values were normalized
to (AGA)10overexpressing a vector control.
Decoding interactions impair translation
not observe a unique band corresponding to the RNA frag-
ment from strains with constructs in which the CGA codons
are near the N-terminus, most likely because the expected
difference in size between the full-length and truncated RNAs
is small (39–60 bases out of >800 bases) (Fig. 6B). Chen et al.
(2010) have reported similar results: they observed an RNA
cleavage product from the PGK1 gene bearing four CGA
codons. Thus, we consider it highly likely that this shorter
mRNA with homology to firefly luciferase is the result of
a ribosome stall at or near the CGA codons.
Adjacent CGA codons are more effective inhibitors
than separated CGA codons
Although the existence of codon pair bias is well docu-
mented, its significance in gene expression has not been
explicitly examined (Smith and Yarus 1989; Irwin et al.
1995; Kane 1995; Moura et al. 2005; Buchan et al. 2006;
Coleman et al. 2008). To elucidate the importance of codon
interactions in modulating expression, we compared the
inhibitory effects of different arrangements of five CGA
and five AGA codons (Fig. 7A,B). Since there was z15-fold
more luciferase from strains in which the construct lacks
any adjacent CGA codons [(CGA–AGA)5] compared to
those with five adjacent CGA codons [(AGA5CGA5) or
(CGA5AGA5)], codon arrangement plays an important role
in CGA-mediated inhibition of expression.
To quantitatively assess the contribution of individual
CGA codons, adjacent codon pairs and alternating codons,
we increased the Arg repeat to 16. This number allowed us to
insert spacers of (AGA)2between various CGA-containing
test sequences, such that CGA codons in adjacent test se-
quences could not reside within a ribosome simultaneously,
and to insert a sufficient number of test sequences to es-
tablish the relationship between expression and CGA dosage
by systematic variation (Fig. 7C).
For all three arrangements of CGA codon test sequences,
there was an exponential dependence of the inhibition of
expression on the number of CGA codons (Fig. 7D–F),
indicating that each test sequence acts independently. For
each series, we derived the exponential coefficient of trans-
lation inhibition, called kTI. In each case, this coefficient
had a very similar value for CGA codons upstream of firefly
or Renilla luciferase (Fig. 7D–F; Table 2).
FIGURE 4. Suppression of CGA-mediated expression defects by an exact base-pairing anticodon mutated tRNAArg(UCG). (A) Effect of indicated
native and anticodon-mutated tRNAArgspecies, expressed on high copy plasmids, on firefly luciferase activity of X4F-reporter constructs
containing (CGA)10insertions. The parent tRNA of each anticodon-mutated tRNAArgis indicated by the first anticodon shown below the figure.
Values were compared to activity of X4F-(AGA)10luciferase in a strain with an empty vector. (B) Effect of an anticodon-mutated tRNAArgspecies,
expressed on a high copy plasmid, on firefly luciferase activity of a RX314F-reporter construct containing a (CGA)10insertion. (C) Suppression of
the inhibitory effects of CGA codons by integrated copies of the mutant tRNAArg(UCG)species that decode CGA without wobble. Luciferase
activity of X4F-reporter constructs containing (Arg)10insertions, specified by (AGA)10, [(CGA)5(AGA)5], or (CGA)10, in yeast strains bearing an
integrated copy of either of two native tRNAArgspecies, two nonnatural exact match tRNAArgspecies, or a vector control as indicated. Two
individual integrant strains were analyzed in each case. Values were normalized to the average of the activity of X4F-(AGA)10luciferase in the two
integrant strains expressing a vector control. Error bars represent the standard deviation from three independent transformants, each assayed in
triplicate. (D) A closer examination of expression of X4F-reporter constructs containing (CGA)10insertions in indicated strains with an expanded
scale of the y-axis. Values are normalized as in C.
Letzring et al.
RNA, Vol. 16, No. 12
Strikingly, CGA–CGA codon pairs were far more effec-
tive inhibitors of expression than isolated CGA codons
or alternating CGA–AGA codon combinations. Whereas
the kTIS(for single isolated codons) was ?0.21 and ?0.25
for the two reporters, the kTIP(for codon pairs) was ?0.53
and ?0.48 for CGA codon pairs, for the two reporters,
respectively (Table 2). Thus, there appears to be a synergis-
tic interaction between codons that occupy the ribosome
simultaneously in adjacent sites. This synergy does not
occur with CGA codons that might occupy the separated A
and E sites, since the kTIAfor alternating (CGA–AGA)
codons was ?0.22, nearly identical to that of isolated CGA
codons (Fig. 7F). We note, however, that even individual
CGA codons were themselves strongly inhibitory, since 10
isolated CGA codons would be expected to yield z14%C
(y = 114e?0.21x, where x = 10) (Fig. 7D).
The major result that emerges from this study is that
decoding of the CGA codon in yeast interferes with an in-
trinsic aspect of the translation elongation reaction. Three
features of the CGA translational inhibitory signal are
notable. First, the critical defect in decoding CGA appears
to be IdA wobble decoding, and not tRNA abundance, since
a single copy of an exact base-pairing mutant tRNAArg(UCG)
efficiently suppressed the expression defects caused by the
CGA codon, whereas an approximate sixfold excess of
tRNAArg(ICG)only mildly suppressed the expression defect.
This means that wobble decoding modulates the efficiency
of elongation, a primary catalytic function of the ribosome.
The inhibition of expression reported here is clearly caused
by translation of the CGA codon, and not by poly-arginine,
since the expression effect is codon-dependent, is suppress-
ible by the anticodon-mutated tRNAArg(UCG), and does not
require long stretches of arginine. Second, although the loss
of expression caused by defects in decoding CGA codons
is not mediated by mRNA degradation, CGA codons do
result in formation of a novel RNA, most likely due to
endonucleolytic cleavage near a stalled ribosome. This means
that translation of sense codons can induce a strong and
efficient halt to translation. Third, adjacent CGA codons
are far more potent inhibitors of expression than separated
CGA codons, a phenomenon most easily ascribed to in-
teractions within the ribosome. Thus, profound differences
in translation efficiency can stem directly from decoding
interactions in the ribosome.
Observations from our work and others provide two
clues to the mechanism by which CGA decoding impairs
translation efficiency. First, the rate of acceptance of the
tRNAArg(ICG)into an A site occupied with a CGA codon may
be slow, since overproduction of the native tRNAArg(ICG)
partially suppressed the CGA-mediated expression defect,
indicating that charged tRNAArg(ICG)is limiting for this
codon. Acceptance into the A site may be slow because of
the altered geometry of the ICG anticodon base-paired with
a CGA codon in the ribosome (Murphy and Ramakrishnan
2004). Second, CGA codons are likely to exert their effects
in two successive steps during translation, since adjacent
CGA codons have a synergistic effect on expression. This
might mean that ribosomes have a memory of the last
decoding reaction in the A site, an idea for which there is no
precedent, or that the IdA wobble interaction is also recog-
nized as defective in the P site. Zaher and Green (2009)
recently identified a quality control pathway in E. coli in
which a mismatch in the P site provokes an increased rate
of misincorporation into the A site, and ultimately results
in premature release. A similar pathway in yeast that probes
the codon–anticodon interaction in the P site would ac-
count for the increased potency of adjacent CGA codons.
Many translation defects are closely coupled to mRNA
degradation. For example, the presence of a premature
FIGURE 5. CGA-mediated inhibition affects firefly luciferase activity
and protein levels, but does not result in reduced luciferase mRNA.
Analysis of firefly luciferase protein, mRNA, and activity in X4F-
reporter constructs containing RGS-(His)6-(Arg)8 insertions, from
yeast strains bearing the indicated constructs (lanes 1–10). Firefly
luciferase protein was detected with antibody to RGS-(His)6, with
antibody to enolase serving as an internal control. In lanes 11–14, the
top two panels contain dilutions of crude extracts expressing the X4F-
RGS-(His)6(AGA)8reporter construct (10, 7.5, 5.0, 2.5 mg). Firefly
luciferase mRNA was measured by RT-PCR analysis using primers
homologous to bases 21–46 and 148–173, as indicated on the diagram
below the panel; sequences are given in Supplemental Table S3;
measurements of Actin mRNA served as an internal control. In the
third panel, lanes 11-14 contain RT-PCR reactions in which dilutions
of DNase-treated RNA from X4F-RGS-(His)6(CGA)8-containing sam-
ple (0.6, 0.3, 0.15, 0.075 mg) were used in the RT reactions, with equal
volumes of the RT reaction in each PCR reaction. Reverse transcrip-
tase reactions in lanes 1–10 contain 0.3 mg total RNA input. Firefly
luciferase mRNA amounts were first normalized to the levels of Actin
mRNA and then quantified relative to the wild-type X4F-reporter
construct with no insertion; the FLuc mRNA levels in the wild-type
strain were arbitrarily set to 100. The presence of RGS-(His)6 is
indicated by an asterisk (*). Firefly luciferase activity of X4F-reporter
constructs containing RGS-(His)6-(Arg)8insertions was normalized
to the wild-type X4F-reporter construct with no insertion.
Decoding interactions impair translation
stop codon results in nonsense-mediated decay (Isken and
Maquat 2007), an RNA structure that blocks ribosome
progress results in no-go decay (Doma and Parker 2006),
and the absence of a stop codon or translation into the
39 UTR results in nonstop decay (Frischmeyer et al. 2002;
van Hoof et al. 2002; Inada and Aiba 2005). It is therefore
not surprising that CGA-mediated inhibition of expression
is associated with cleavage of the mRNA at or near the CGA
codons, although it is surprising that the resulting 39 frag-
ment appears to be stable, since there is a large amount of
it. We infer that ribosomes arrest at CGA codons, since
CGA codons in two contexts result in a new RNA with
properties expected of an endonucleolytic cleavage product
near the CGA codons (our data; as well as Chen et al.
2010). The ribosome may remain bound to the 59 end of
the fragment either because the arginine residues in the exit
tunnel hold it there (Dimitrova et al. 2009), or because the
ribosome with both the A and P sites occupied with
tRNAArg(ICG)is arrested but left in a translation competent
mode. Tuller et al. (2010a) noted that clusters of poorly
adapted codons are enriched within the first 50 codons, and
proposed that such clusters function to stall ribosomes
FIGURE 6. The effects of CGA codons at amino acids 4 and 314 on full-length mRNA, examined by Northern analysis. (A) Diagram of the X4R
Renilla luciferase reporter indicating the position of the oligonucleotide probe used in panels B–D. Probe c is located at base pairs 682–705 in the
935 base-pair Renilla luciferase coding sequence. (B) Analysis of the effects of insertion of CGA codons at amino acid 4 upstream of Renilla
luciferase on Renilla luciferase mRNA and activity. Renilla luciferase mRNA from X4R reporter constructs containing RGS-(His)6-(Arg)8was
examined by Northern blots with the probe c indicated in panel A; U2 snRNA served as an internal control. Renilla luciferase mRNA levels were
first normalized to levels of U2 snRNA and then compared to the wild-type Renilla luciferase mRNA containing RGS-(His)6. In the lower panel,
luciferase activity of Renilla luciferase reporter constructs containing RGS-(His)6-(Arg)8insertions are shown. (Off, expression under repressing
conditions.) Lanes 1–4 contain different amounts of input total RNA from the X4R- RGS-(His)6(CGA)8bearing strain (20, 10, 5, 2.5 mg), while
lanes 5–12 contain RNA (20 mg) from yeast strains bearing the indicated constructs. (C) Decoding of CGA codons with an anticodon-mutated
tRNAArgspecies suppresses the CGA-mediated increase in Renilla luciferase mRNA. Northern blot analysis of Renilla luciferase mRNA from
Renilla luciferase reporter constructs containing RGS-(His)6-(Arg)8, using Actin as an internal control. Strains expressed either a nonnatural exact
match tRNA or a vector control. Luciferase mRNA amounts were normalized to the levels of Actin mRNA and then to RGS-(His)6(AGA)8; In the
titration indicated with the triangle, mRNA input from the strain X4R- RGS-(His)6(CGA)8in a vector control was varied (20, 10, 5 mg). (D)
Insertion of stop codons at amino acid 4 of Renilla luciferase resulted in a reduction in stable Renilla luciferase mRNA. Northern blot analysis of
Renilla luciferase mRNA from indicated Renilla luciferase reporter constructs. Renilla luciferase mRNA was first normalized to levels of U1
snRNA and then to RLuc (wt) mRNA. (E) A diagram of the RX314F reporter indicating the relative position of the probes used in panel F. Probe c
is located at base pairs 682–705 in Renilla luciferase (935 base pairs), while probe d is located at base pairs 1564–1593 in the RX314F reporter (2631
base pairs); this corresponds to base pairs 601–630 in firefly luciferase (1668 base pairs). Probe sequences are shown in Supplemental Table S3. (F)
Analysis of the effects of insertion of CGA codons at amino acid 314 in the RX314F reporter on Renilla-firefly luciferase mRNA. RNA from strains
bearing RX314F constructs with RGS-(His)6,or RGS-(His)6-(Arg)8encoded with either (AGA)8, [(CGA)4(AGA)4], or (CGA)8as indicated was
examined by Northern blots with the probes indicated in panel E. U2 snRNA served as an internal control.
Letzring et al.
RNA, Vol. 16, No. 12
early in translation to prevent collisions between elongat-
ing ribosomes. If rare codons do play a role in mediating
ribosome density, then mechanisms may have evolved to
prevent mRNA decay in response to codon-mediated ribo-
Although codon choice has been linked for many years to
the large disparities in translation efficiency (Grantham et al.
1980; Bennetzen and Hall 1982; Ikemura 1982; Duret and
Mouchiroud 1999; Ghaemmaghami et al. 2003; Brockmann
et al. 2007), we still do not know the extent to which codon
choice directly influences translation efficiency, or the iden-
tity and properties of all of the codon-initiated signals,
particularly in eukaryotes. With this systematic analysis, we
found that most codon repeats have only small effects on
expression, making the CGA codon conspicuous in its
effects. Given that CGA codon pairs are substantially more
effective inhibitors of expression than isolated CGA co-
dons, it is likely that other codon combinations, that are
more potent inhibitors of expression than the individual
codon repeats, remain to be discovered.
Three lines of reasoning support the idea that codon-
mediated regulation of expression is functionally important
and nearly universal. First, IdA wobble decoding of CGA is
conserved in the hemiascomycetes fungi, as well as in many
bacteria (Curran 1995; Grosjean et al. 2010), even though it
is presumed to be inefficient, as described here. Although
CGA is the only codon in yeast decoded with an IdA wobble
interaction (Johansson et al. 2008), it is highly unlikely that
FIGURE 7. CGA codon pairs are more inhibitory than single CGA codons. (A,B) The effects of the arrangement of five CGA codons within an
(Arg)10insert on Renilla luciferase activity were compared to each other and to Renilla luciferase activity from constructs containing (Arg)10
insertions specified by (AGA)10or (CGA)10. (A) The arrangements of Arg CGA codons with the number of CGA codons and codon pairs
indicated and (B) the resulting luciferase activity with lanes corresponding to constructs in A. Reported values were corrected to an independently
transcribed firefly luciferase gene, and then normalized to (AGA)10. (C) Schematic of the arrangement of CGA codons in test constructs designed
to examine the effects of single CGA codons, pairs of CGA codons, and alternating CGA codons. (D) Relative luciferase activity of firefly luciferase
reporter constructs containing (Arg)16insertions with increasing CGA content arranged either as single codons or codon pairs. Reported values
were corrected to an independently transcribed Renilla luciferase gene, and then normalized to (AGA)16. (E) Relative luciferase activity of Renilla
luciferase reporter constructs containing (Arg)16insertions with increasing CGA content arranged either as single codons or codon pairs.
Reported values were corrected to an independently transcribed firefly luciferase gene, and then normalized to (AGA)16. (F) Relative luciferase
activity of Renilla luciferase reporter constructs containing (Arg)16insertions with increasing CGA content. CGA codons are clustered either as
single codons separated by two AGA codons or as alternating codons (CGA–AGA). Reported values were corrected to an independently
transcribed firefly luciferase gene, and then normalized to (AGA)16.
Decoding interactions impair translation
widespread retention of this decoding strategy is accidental.
Since expression of the exact match tRNAArg(UCG)does not
result in obvious growth defects on either minimal or rich
plates at temperatures from 18°C to 37°C (data not shown),
such mutations have undoubtedly occurred in nature and
been discarded. Presumably the retention of this inefficient
decoding has an important, but as yet undiscovered, role.
Second, organisms go to great lengths to vary the decoding
efficiency at different codons. All organisms use 49 (or
fewer) tRNAs to decode the 61 sense codons, making
wobble decoding of some codons obligatory. In many
organisms, there is tremendous variation in the amounts
of tRNAs that decode particular codons, as exemplified by
the one to 17 copies of different tRNA genes in yeast
(Grosjean et al. 2010). In addition, the sequences of tRNAs
that all decode the same codon (isodecoders) vary. In
humans, the 49 isoaccepting tRNA families are encoded by
450 tRNA genes, which include z270 isodecoders, some of
which appear to be functionally distinct (Geslain and Pan
2010). Third, reduced translation efficiency of some genes
may be under selective pressure. Even in human genes,
clusters of codons decoded by rare codons are found more
frequently than expected, are frequently conserved between
human and chimp orthologs, and sometimes cluster into
distinct functional groups (Neafsey and Galagan 2007;
Parmley and Huynen 2009). A resolution to these issues
of codon usage requires further identification of the precise
codon combinations that reduce translation efficiency, and
an analysis of their distribution in genomes and functional
MATERIALS AND METHODS
Plasmids and strains
Both X4F-firefly luciferase (pDL306) and RX314F dual luciferase
(pDL202) constructs were constructed from dual luciferase vector
pDB688 (Keeling et al. 2004) and contain unique SalI and BamHI
sites for codon insertions. To construct X4F (pDL306), the Renilla
luciferase gene was removed by BamHI digestion followed by
ligation of oligonucleotides DLO188 and DLO189 containing an
ATG start codon (Supplemental Table S3). To construct RX314F
(pDL202), the BamHI site upstream of the Renilla luciferase gene
was mutated by PCR amplification of pDB688 with DLO99 and
DLO100 (Supplemental Table S3). Vectors
(JP0054 and JP0056), in which Renilla and
firefly luciferase genes were expressed under
PGAL1,10control, were constructed by indi-
vidual PCR amplification of the firefly and
Renilla luciferase genes with additional se-
quences required for ligation-independent
cloning into a 2m URA3 vector BG2596,
which is derived from BG2483 by insertion of
sequences containing PGAL10and the CYC1
transcription terminator (details available
upon request) (Malkowski et al. 2007).
Unique NheI and BamHI sites were inserted
in firefly luciferase (JP0054) or Renilla luciferase (JP0056). Oli-
gonucleotides containing codon insertions (Supplemental Table
S3) were annealed and ligated into SalI-BamHI or NheI-BamHI
digested vectors, which were transformed into E. coli (XL1-Blue)
and confirmed by sequence analysis. Vector pEKD1024 carries the
superfolder GFP (Pedelacq et al. 2006) and yeast optimized RFP
(Keppler-Ross et al. 2008) under control of the bidirectional
GAL1,10 promoter inserted into integrating vector JW132
(J Whipple and E Phizicky, unpubl.).
To construct 2m plasmids containing tRNAArgspecies with their
own promoters, tRNAs tR(CCG)L, tR(CCU)J, tR(UCU)K, and
tR(ACG)D, with z300 base pairs of upstream and downstream
sequences, were amplified by PCR from yeast genomic DNA with
oligonucleotides listed in Supplemental Table S3. Purified PCR
products were cloned into 2m vector AVA0577 (Alexandrov et al.
2006) by standard ligation-independent cloning (LIC) (Aslanidis
and de Jong 1990; Alexandrov et al. 2004). To construct the
anticodon mutated exact base-pairing tRNAArgvariants, pDL866
(tR(UCU)K) and pDL867 (tR(ACG)D) were subjected to site-
directed mutagenesis with a Quickchange kit (Stratagene 200518)
according to the manufacturer’s instructions using oligonucleotides
DLO444, DLO445, DLO508, and DLO509 (Supplemental Table
S3). To integrate tRNAArgvariants, tRNA genes from pDL866
(tR(UCU)K), pDL867 (tR(ACG)D), pDL869 (tR(UCU-U35G)),
and pDL870 (tR(ACG-A34U)) were amplified by PCR with
oligonucleotides DLO575 and DLO576, ligated into integrating
vector JW132 by standard LIC cloning procedures (Alexandrov
et al. 2004). A linear fragment containing the tRNAArgvariants
was produced by restriction digestion and transformed into yeast
strain BY4741. Yeast strains, derived from BY4741 (MATa his3D1,
leu2D0, met15D0, ura3D0), are listed in Supplemental Table S1
and plasmids used in these studies are listed in Supplemental
Transformants were grown in SD-uracil or SC-uracil with 2%
raffinose, 2% galactose media (Sherman et al. 1986) to OD600be-
tween 0.8 and 1.0. Cells (0.8 OD-mL) were harvested by centrifu-
gation at 4000 rpm, washed with cold ddH2O, and resuspended in
1X Passive Lysis Buffer (100 mL) (Promega), nutated for 15 min at
25°C, followed by incubation at 25°C for 20 min., then 2 mL lysate
was added to 20 mL Luciferase Assay Reagent II (Dual Luciferase
Reporter Assay System, Promega PAE1910). Firefly luciferase
activity was measured in Relative Luminescence Units (RLUs),
with a 2-sec delay and a 10-sec measurement using a Berthold
FB12 luminometer. To assay Renilla luciferase activity in the same
TABLE 2. Exponential coefficient of translation inhibition, kTIvalues, for CGA in different
Test arrangement LayoutReporterkTI
*AGA AGA (CGA?AGA?AGA)xAGA(14-3x)
CGA pairs (CGA?CGA?AGA?AGA)xAGA(16-4x)
AGA AGA (CGA?AGA)xAGA(14-2x)
Letzring et al.
RNA, Vol. 16, No. 12
reaction, 20 mL 1X Stop and Glo (Promega) was added, and
activity was measured as described above. In the systematic anal-
ysis of codon effects, activity was measured from three indepen-
dent transformants, and three readings were performed on each
lysate. Error bars reflect the standard deviation between three
Protein expression and RNA analysis
Yeast transformants, in which Renilla and firefly luciferase genes
were under control of the GAL1,10 promoter, were grown as
described (Quartley et al. 2009) except that strains were grown
overnight in raffinose to an OD600 between 1.8 and 2.4, and
expression was induced by addition of 3X YP media with 6%
galactose (1/3 final volume), followed by growth for 6 h. Crude
extract preparation and Western blot analysis were performed as
described (Quartley et al. 2009). Membranes were incubated
either with mouse anti-RGS-(His)6 (1:2500 dilution) or with
rabbit anti-enolase (1:25,000 dilution), followed by washing as
described previously, and incubation with either HRP-conjugated
Goat IgG anti-mouse (1:10,000 dilution, BioRad) for anti-RGS-
(His)6or HRP-conjugated Goat IgG anti-rabbit (1:10,000 dilu-
tion, Biorad) for anti-enolase, and development with ECL Plus
Western blotting detection system (GE Healthcare RPN2132).
RNA for RT-PCR was prepared from 50 OD-mL cells as
described (Schmitt et al. 1990). RNA (5 mg) was treated with
1.25 U RNase free RQ DNase I (Promega M6101), followed by
cDNA synthesis from DNase-treated RNA (0.3 mg) using Moloney
murine leukemia virus reverse transcriptase (Invitrogen); the
resulting cDNA was amplified by PCR using primers DLO527,
DLO528, DLO531, and DLO532 (Supplemental Table S3).
Expression of green fluorescent protein in yeast strains bearing
an integrated copy of pEKD1163, pEKD1170, or pEKD1167 was
assessed after growth in YP + adenine + 2% galactose + 2%
raffinose. Cells were washed in cold ddH2O, collected by centri-
fugation, resuspended in 1X PBS, and subjected to analysis on
the FACSCantoII with excitation at 488 nM and detection of
fluorescence emission using a 530/30 band pass filter.
For analysis by Northern blots, yeast transformants (150 OD-
mL), grown as described above, were resuspended in 3 mL RNA
isobuffer (0.5 M NaCl, 0.2 M Tris-Cl, pH 7.5, 0.01 M EDTA, 1%
SDS) followed by addition of 3 mL phenol-chloroform-isoamyl
alcohol (PCA) (25:24:1), equilibrated at pH 7.5, and glass beads,
and lysed by vortexing 10 times for 20 sec with 1 min on ice
between cycles. Additional RNA isobuffer (4.5 mL) and PCA
(4.5 mL) were added, and lysates were centrifugated; the aqueous
phase was re-extracted with 4.5mL PCA, nucleic acids were pre-
cipitated with ethanol, and the pellet was resuspended in TE,
pH 7.5, and stored at ?80°C. Total RNA (20 mg) was separated
by electrophoresis either in a 1.5% formaldehyde agarose gel
(Sambrook et al. 1989) or in a 6% polyacrylamide gel (19:1)
containing 7 M urea, transferred to Genescreen plus membrane,
and UV crosslinked to the membrane at 254 nm for 2 min. mRNA
was visualized by synthetic oligonucleotide probes DLO511,
DLO531, and SHO21 (Supplemental Table S3) labeled at the
59 end with
32P using T4 polynucleotide kinase (Roche).
Supplemental material can be found at http://www.rnajournal.org.
We thank Eric Phizicky, Stan Fields, Gloria Culver, Roy Parker,
and Mark Dumont for advice during the course of the work and
for comments on the manuscript, and Joe Whipple and E.P. for
the integrating vector. This work was supported by NSF grant no.
MCB-0919658 awarded to E.J.G.
Received August 12, 2010; accepted September 24, 2010.
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